Reviewer #2 (Public Review):

This work focuses on the biochemical features of the SARS-CoV-2 Nucleocapsid (N) protein, which condenses the large viral RNA genome inside the virus and also plays other roles in the infected cell. The N protein of SARS-CoV-2 and other coronaviruses is known to contain two globular RNA-binding domains, the NTD and CTD, flanked by disordered regions. The central disordered linker is particularly well understood: it contains a long SR-rich region that is extensively phosphorylated in infected cells, followed by a leucine-rich helical segment that was shown previously by these authors to promote N protein oligomerization.

In the current work, the authors analyze 5 million viral sequence variants to assess the conservation of specific amino acids and general sequence features in the major regions of the N protein. This analysis shows that disordered regions are particularly variable but that the general hydrophobic and charge character of these regions are conserved, particularly in the SR and leucine-rich regions of the central linker. The authors then construct a series of N proteins bearing the most prevalent mutations seen in the Delta and Omicron variants, and they subject these mutant proteins to a comprehensive array of biophysical analyses (temperature sensitivity, circular dichroism, oligomerization, RNA binding, and phase separation).

The results include a number of novel findings that are worthy of further exploration. Most notable are the analyses of the previously unstudied P31L mutation of the Omicron variant. The authors use ColabFold and sedimentation analysis to suggest that this mutation promotes self-association of the disordered N-terminal region and stimulates the formation of N protein condensates. Although the affinity of this interaction is low, it seems likely that this mutation enhances viral fitness by promoting N-terminal interactions. The work also addresses the impact of another unstudied mutation, D63G, that is located on the surface of the globular NTD and has no significant effect on the properties analyzed here, raising interesting questions about how this mutation enhances viral fitness. Finally, the paper ends with studies showing that another common mutant, R203K/G204R, disrupts phase separation and might thereby alter N protein function in a way that enhances viral fitness. These provocative results set the stage for in-depth analyses of these mutations in future work.

Reviewer #1 (Public Review):

Summary:

The authors were using an innovative technic to study the visual vigilance based on high-acuity vision, the fovea. Combining motion-capture features and visual space around the head, the authors were able to estimate the visual fixation of free-feeding pigeon at any moment. Simulating predator attacks on screens, they showed that 1) pigeons used their fovea to inspect predators cues, 2) the behavioural state (feeding or head-up) influenced the latency to use the fovea and 3) the use of the fovea decrease the latency to escape of both the individual that foveate the predators cues but also the other flock members.

Strengths:

The paper is very interesting, and combines innovative technic well adapted to study the importance of high-acuity vision for spotting a predator, but also of improving the behavioural response (escaping). The results are strong and the models used are well-adapted. This paper is a major contribution to our understanding of the use of visual adaptation in a foraging context when at risk. This is also a major contribution to the understanding of individual interaction in a flock.

Weaknesses:

I have identified only two weaknesses:

(1) The authors often mixed the methods and the results, Which reduces the readability and fluidity of the manuscript. I would recommend the authors to re-structure the manuscript.<br /> (2) In some parts, the authors stated that they reconstructed the visual field of the pigeon, which is not true. They identified the foveal positions, but not the visual fields, which involve different sectors (binocular, monocular or blind). Similarly, they sometimes mix-up the area centralis and the fovea, which are two different visual adaptations.

Reviewer #1 (Public Review):

Summary:

Plasmacytoid dendritic cells (pDCs) represent a specialized subset of dendritic cells (DCs) known for their role in producing type I interferons (IFN-I) in response to viral infections. It was believed that pDCs originated from common DC progenitors (CDP). However, recent studies by Rodrigues et al. (Nature Immunology, 2018) and Dress et al. (Nature Immunology, 2019) have challenged this perspective, proposing that pDCs predominantly develop from lymphoid progenitors expressing IL-7R and Ly6D. A minor subset of pDCs arising from CDP has also been identified as functionally distinct, exhibiting reduced IFN-I production but a strong capability to activate T-cell responses. On the other hand, clonal lineage tracing experiments, as recently reported by Feng et al. (Immunity, 2022), have demonstrated a shared origin between pDCs and conventional DCs (cDCs), suggesting a contribution of common DC precursors to the pDC lineage.

In this context, Araujo et al. investigated the heterogeneity of pDCs in terms of both development and function. Their findings revealed that approximately 20% of pDCs originate from lymphoid progenitors common to B cells. Using Mb1-Cre x Bcl11a floxed mice, the authors demonstrated that the development of this subset of pDCs, referred to as "B-pDCs," relied on the transcription factor BCL11a. Functionally, B-pDCs exhibited a diminished capacity to produce IFN-I in response to TLR9 agonists but secreted more IL-12 compared to conventional pDCs. Moreover, B-pDCs, either spontaneously or upon activation, exhibited increased expression of activation markers (CD80/CD86/MHC-II) and a heightened ability to activate T-cell responses in vitro compared to conventional pDCs. Finally, Araujo et al. characterized these B-pDCs at the transcriptomic level using bulk and single-cell RNA sequencing, revealing them as a unique subset of pDCs expressing certain B cell markers such as Mb1, as well as specific markers (Axl) associated with cells recently described as transitional DCs.

Thus, in contrast to previous findings, this study posits that a small proportion of pDCs derive from B cell-committed lymphoid progenitors, and this subset of B-pDCs exhibits distinct functional characteristics, being less specialized in IFN-I production but rather in T cell activation.

Strengths:

Previously, the same research group delineated the significance of BCL11a as a critical transcription factor in pDC development (Ippolito et al., PNAS, 2014). This study elucidates the precise stage during hematopoiesis at which BCL11a expression becomes essential for the emergence of a distinct subset of pDCs, substantiated by robust genetic evidence in vivo. Furthermore, it underscores the shared developmental origin between pDCs and B cells, reinforcing prior research in the field that suggests a lymphoid origin of pDCs. Finally, this work attributes specific functional properties to pDCs originating from these lymphoid progenitors shared with B cells, emphasizing the early imprinting of functional heterogeneity during their development.

Weaknesses:

The authors delineate a subset of pDCs dependent on the BCL11a transcription factor, originating from lymphoid progenitors, and compare it to conventional pDCs, which they suggest differentiate from common DC progenitors of myeloid origin. However, this interpretation lacks support from the authors' data. Their single-cell RNA sequencing data identifies cells corresponding to progenitors (Prog2), from which the majority of pDCs, termed conventional pDCs, likely originate. This progenitor cell population expresses Il7r, Siglech, and Ly6D, but not Csfr1. The authors describe this progenitor as resembling a "pro-pDC myeloid precursor," yet these cells align more closely with lymphoid (Il7r+) progenitors described by Rodrigues et al. (Nature Immunology, 2018) and Dress et al. (Nature Immunology, 2019). Furthermore, analysis of their Mb1 reporter mice reveals that only a fraction of common lymphoid progenitors (CLP) express YFP, giving rise to a fraction of YFP+ pDCs. However, this does not exclude the possibility that YFP- CLP could also give rise to pDCs. The authors could address this caveat by attempting to differentiate pDCs from both YFP+ and YFP- CLPs in vitro in the presence of FLT3L. Additionally, transfer experiments using these lymphoid progenitors could be conducted in vivo to assess their differentiation potential in competitive settings.

Using their Mb1-reporter mice, the authors demonstrate that YFP pDCs originating from lymphoid progenitors are functionally distinct from conventional pDCs, mostly in vitro, but their in vivo relevance remains unknown. It is crucial to investigate how Bcl11a conditional deficiency in Mb1-expressing cells affects the anti-viral immune response, for example, using the M-CoV infection model as described by Sulczewski et al. in Nature Immunology, 2023. Particularly, the authors suggest that their B-pDCs act as antigen-presenting cells involved in T-cell activation compared to conventional pDCs. However, these findings contrast with those of Rodrigues et al., who have shown that pDCs of myeloid origin are more effective than pDCs of lymphoid origin in activating T-cell responses. The authors should discuss these discrepancies in greater detail. It is also notable that B-PDCs acquire the expression of ID2 (Figure S3A), commonly a marker of conventional/myeloid DCs. The authors could analyze in more detail the acquisition of specific myeloid features (CD11c, CX3CR1) by this B-PDCs subset and discuss how the expression of ID2 may impair classical pDC features, as ID2 is a repressor of E2-2, a master regulator of pDC fate.

Finally, through the analysis of their single-cell RNA sequencing data, the authors show that the subset of B-pDCs they identified expresses Axl, confirmed at the protein level. Given this specific expression profile, the authors suggest that B-pDCs are related to a previously described subset of transitional DCs, which were reported to share a common developmental path with pDCs, (Sulczewski et al. in Nature Immunology, 2023). While intriguing, this observation requires further phenotypic and functional characterization to substantiate this claim.

Reviewer #1 (Public Review):

Summary:

C. elegans NHL-2 is a member of the conserved TRIM-NHL RNA binding protein family, with known functions in promoting small regulatory RNA function, including the conserved let-7 family microRNAs. Since NHL-2 promotes microRNA function, the authors seek to address if this function is due to direct binding of a mRNA target shared with the miRNA pathway. They successfully solve the crystal structure of NHL-2's NHL domain and discover residues Tyr935/Arg978 are required for RNA binding in vitro. In C. elegans, they establish that Tyr935/Arg978 are required for nhl-2 to promote let-7 microRNA function. Processing body (P body) size is increased in nhl-2 (Y935A R978A) and null mutants. The microRNA Argonautes, ALG-1 and ALG-2, also show increased binding to known let-7 mRNA targets in nhl-2 null mutants. Together these data suggest a lack of mRNA turnover in the absence of functional NHL-2. NHL-2 may function with CGH-1 and IFET-1 to promote let-7 miRISC function.

Strengths:

The authors successfully solve the structure of NHL-2's NHL domain. Although unable to crystalize it bound to RNA they are able to predict residues important for RNA binding based on charge, position and comparison with other known NHL domain structures crystalized with RNA. In vitro RNA binding assays confirm that Tyr935/Arg978 are required for RNA binding in vitro.

Weaknesses:

(1) In vivo, authors use a combination of established let-7 microRNA genetics and a 3' UTR reporter assay to establish that Tyr935/Arg978 are required for nhl-2 to promote let-7 microRNA function. However, they do not demonstrate that full length NHL-2 actually binds RNA directly in vivo in the Tyr935/Arg978 mutated background. While the presented genetic evidence suggests nhl(RBlf) acts much like the nhl-2 null, it is never demonstrated that full length NHL-2(RBlf) is actually RNA binding defective/dead in vivo. Yet several times in the text this is implied or stated. For example,<br /> o page 8, section title. "RNA binding is essential for NHL-2 function in heterochronic pathway"<br /> o page 9 - line 13-14. "Together, these data indicate that the RING and NHL domains are required for the normal function of NHL-2, but that the loss of RNA-binding activity has a more pronounced phenotype, suggesting that RNA-binding is critical for NHL-2 function."<br /> o page 11, line 3-4. "Together these experiments support the conclusion that... RNA binding is essential for its function"<br /> The language should be softened (e.g., page 8: "Residues required for RNA binding in vitro are required for NHL-2 function in heterochronic path") or additional experiments should be performed to support that NHL-2(RBlf) is in fact RNA binding defective/dead, like wild-type NHL-2 vs NHL-2(RBlf) RIP-qPCR for let-7 targets.

(2) Authors report that Processing body (P body) size is dependent on nhl-2 and the Tyr935/Arg978 residues. microRNA Argonautes, ALG-1 and ALG-2, also show increased binding to known let-7 mRNA targets in nhl-2 null mutants (unfortunately requirement of Tyr935/Arg978 is not tested). However total levels of these mRNAs are unchanged. Authors propose these data together support a role for nhl-2 in promoting microRNA target turnover. Unfortunately, it is unclear how increased P body size with no observed increase of microRNA target levels are to be resolved.

(3) The authors propose a model where NHL-2, CGH-1(DDX6) and IFET-1(eIF4E-transporter/4E-T) promote microRNA mediated translational repression and possibly turnover based on nhl-2-dependent IFET-1 interaction with ALG-1, cgh-1's synthetic interaction with both nhl-2 and ifet-1 to enhance let-7-mediated alae development, and conservation of known interactions between Dead Box helicases and eiF4A, which is supplemented by ALPHAFold modelling of IFET-1. The Boag lab previously characterized ifet-1 as a translational repressor required for germline P granule formation (Sengupta 2013 J Cell Sci). The role of NHL-2 RNA binding is unclear in this model as is any more molecular evidence of direct NHL-2, CGH-1 and IFET-1 interaction.

(4) In Figure 5, adult nhl-2(ok818) worms express the mCherry when the putative NHL-2 binding sites in the lin-28 3'UTR reporter are mutated. Couldn't this be interpreted as suggesting that the observed phenotype is nhl-2 independent? The authors mention this as an "interesting" observation in text, but I find it concerning. The authors should address this issue more directly. The reporter expression data needs to be quantified.

(5) I am frankly confused at the direction the manuscript takes in the Discussion section. The role of NHL-2 RNA binding, which has been the core of the paper, is seemingly disregarded and exchanged for what is mainly speculation about protein-protein level regulation with CGH-1 and IFET-1. This is all based on only a few pieces of data that do not include any analysis using the nhl-2(RBlf): nhl-2-dependent IFET-1 interaction with ALG-1, cgh-1's synthetic interaction with both nhl-2 and ifet-1 to enhance let-7-mediated alae development, and conservation of known interactions between Dead Box helicases and eiF4A, which is supplemented by ALPHAFold modelling of IFET-1. I'd strongly suggest reworking the text to better integrate IFET-1 or skip it and refocus the Discussion around the majority of the data characterizing NHL-2 RNA binding.

Reviewer #1 (Public Review):

Summary:

The "optorepressilator", an optically controllable genetic oscillator based on the famous E. coli 3-repressor (LacI, TetR, CI) oscillator "repressilator", was developed. An individual repressilator shows a stable oscillation of the protein levels with a relatively long period that extends a few doubling times of E. coli, but when many cells oscillate, their phases tend to desynchronize. The authors introduced an additional optically controllable promoter through a conformal change of CcaS protein and let it control how much additional CI is produced. By tightly controlling the leak from the added promoter, the authors successfully kept the original repressilator oscillation when the added promoter was not activated. In contrast, the oscillation was stopped by expressing the additional CI. Using this system, the authors showed that it is possible to synchronise the phase of the oscillation, especially when the activation happens as a short pulse at the right phase of the repressilator oscillation. The authors further show that, by changing the frequency of the short pulses, the repressilator was entrained to various ratios to the pulse period, and the author could reconstruct the so-called "Arnold tongues", the signature of entrainment of the nonlinear oscillator to externally added periodic perturbation. The behaviour is consistent with the simplified mathematical model that simulates the protein concentration using ordinary differential equations.

Strengths:

Optical control of the oscillation of the protein clock is a powerful and clean tool for studying the synthetic oscillator's response to perturbation in a well-controlled and tunable manner. The article utilizes the plate reader setup for population average measurements and the mother machine setup for single-cell measurements, and they compensate nicely to acquire necessary information.

Weaknesses:

The current paper added the optogenetically controlled perturbation to control the phase of oscillation and entrainment, but there are a few other works that add external perturbation to a collection of cells that individually oscillate to study phase shift and/or entrainment. The current paper lacks discussion about the pros and cons of the current system compared to previously analyzed systems.

Reviewer #1 (Public Review):

The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth considering and exploring further.

After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revision. However, one piece of new data is perplexing to me. The new Figure 7 presents the results of a model analysis of the strength of the EI caused by a second fish to localize when the focal fish is chirping. From my understanding of this type of model, EOD frequency is not a parameter in the model since it evaluates the strength of the field at a given point in time. Therefore the only thing that matters is the phase relationship and strength of the EOD. Assuming that the second fish's EOD is kept constant and the phases relationship is also the same, the only difference during a chirp that could affect the result of the calculation is the potential decrease in EOD amplitude during the chirp. It is indeed logical that if the focal fish decreased its EOD amplitude the target fish's EOD becomes relatively stronger. Where things are harder to understand is why the different types of chirps (e.g. type 1 vs type 2) lead to the same increase in signal even though they are typically associated with different levels of amplitude modulations. Also, it is hard to imagine that a type 2 chirps that is barely associated with any decrease in EOD amplitude (0-10% maybe), would cause doubling of the EI strength. There might be something I don't understand but the authors should provide a lot more details on how this result is obtained and convince us that it makes sense.

Reviewer #1 (Public Review):

Summary:

The authors present tviblindi, a computational workflow for trajectory inference from molecular data at single-cell resolution. The method is based on (i) pseudo-time inference via expecting hitting time, (ii) sampling of random walks in a directed acyclic k-NN where edges are oriented away from a cell of origin w.r.t. the involved nodes' expected hitting times, and (iii) clustering of the random walks via persistent homology. An extended use case on mass cytometry data shows that tviblindi can be used elucidate the biology of T cell development.

Strengths:

- Overall, the paper is very well written and most (but not all, see below) steps of the tviblindi algorithm are explained well.

- The T cell biology use case is convincing (at least to me: I'm not an immunologist, only a bioinformatician with a strong interest in immunology).

Weaknesses:

- The main weakness of the paper is that a systematic comparison of tviblindi against other tools for trajectory inference (there are many) is entirely missing. Even though I really like the algorithmic approach underlying tviblindi, I would therefore not recommend to our wet-lab collaborators that they should use tviblindi to analyze their data. The only validation in the manuscript is the T cell development use case. Although this use case is convincing, it does not suffice for showing that the algorithms's results are systematically trustworthy and more meaningful (at least in some dimension) than trajectories inferred with one of the many existing methods.

- The authors' explanation of the random walk clustering via persistent homology in the Results (subsection "Real-time topological interactive clustering") is not detailed enough, essentially only concept dropping. What does "sparse regions" mean here and what does it mean that "persistent homology" is used? The authors should try to better describe this step such that the reader has a chance to get an intuition how the random walk clustering actually works. This is especially important because the selection of sparse regions is done interactively. Therefore, it's crucial that the users understand how this selection affects the results. For this, the authors must manage to provide a better intuition of the maths behind clustering of random walks via persistent homology.

- To motivate their work, the authors write in the introduction that "TI methods often use multiple steps of dimensionality reduction and/or clustering, inadvertently introducing bias. The choice of hyperparameters also fixes the a priori resolution in a way that is difficult to predict." They claim that tviblindi is better than the original methods because "analysis is performed in the original high-dimensional space, avoiding artifacts of dimensionality reduction." However, in the manuscript, tviblindi is tested only on mass cytometry data which has a much lower dimensionality than scRNA-seq data for which most existing trajectory inference methods are designed. Since tviblindi works on a k-NN graph representation of the input data, it is unclear if it could be run on scRNA-seq data without prior dimensionality reduction. For this, cell-cell distances would have to be computed in the original high-dimensional space, which is problematic due to the very high dimensionality of scRNA-seq data. Of course, the authors could explicitly reduce the scope of tviblindi to data of lower dimensionality, but this would have to be stated explicitly.

- Also tviblindi has at least one hyper-parameter, the number k used to construct the k-NN graphs (there are probably more hidden in the algorithm's subroutines). I did not find a systematic evaluation of the effect of this hyper-parameter.

Reviewer #1 (Public Review):

Summary:

Meissner-Bernard et al present a biologically constrained model of telencephalic area of adult zebrafish, a homologous area to the piriform cortex, and argue for the role of precisely balanced memory networks in olfactory processing.

This is interesting as it can add to recent evidence on the presence of functional subnetworks in multiple sensory cortices. It is also important in deviating from traditional accounts of memory systems as attractor networks. Evidence for attractor networks has been found in some systems, like in the head direction circuits in the flies. However, the presence of attractor dynamics in other modalities, like sensory systems, and their role in computation has been more contentious. This work contributes to this active line of research in experimental and computational neuroscience by suggesting that, rather than being represented in attractor networks and persistent activity, olfactory memories might be coded by balanced excitation-inhibitory subnetworks.

Strengths:

The main strength of the work is in: (1) direct link to biological parameters and measurements, (2) good controls and quantification of the results, and (3) comparison across multiple models.

(1) The authors have done a good job of gathering the current experimental information to inform a biological-constrained spiking model of the telencephalic area of adult zebrafish. The results are compared to previous experimental measurements to choose the right regimes of operation.<br /> (2) Multiple quantification metrics and controls are used to support the main conclusions and to ensure that the key parameters are controlled for - e.g. when comparing across multiple models.<br /> (3) Four specific models (random, scaled I / attractor, and two variant of specific E-I networks - tuned I and tuned E+I) are compared with different metrics, helping to pinpoint which features emerge in which model.

Weaknesses:

Major problems with the work are: (1) mechanistic explanation of the results in specific E-I networks, (2) parameter exploration, and (3) the functional significance of the specific E-I model.

(1) The main problem with the paper is a lack of mechanistic analysis of the models. The models are treated like biological entities and only tested with different assays and metrics to describe their different features (e.g. different geometry of representation in Fig. 4). Given that all the key parameters of the models are known and can be changed (unlike biological networks), it is expected to provide a more analytical account of why specific networks show the reported results. For instance, what is the key mechanism for medium amplification in specific E/I network models (Fig. 3)? How does the specific geometry of representation/manifolds (in Fig. 4) emerge in terms of excitatory-inhibitory interactions, and what are the main mechanisms/parameters? Mechanistic account and analysis of these results are missing in the current version of the paper.

(2) The second major issue with the study is a lack of systematic exploration and analysis of the parameter space. Some parameters are biologically constrained, but not all the parameters. For instance, it is not clear what the justification for the choice of synaptic time scales are (with E synaptic time constants being larger than inhibition: tau_syn_i = 10 ms, tau_syn_E = 30 ms). How would the results change if they are varying these - and other unconstrained - parameters? It is important to show how the main results, especially the manifold localisation, would change by doing a systematic exploration of the key parameters and performing some sensitivity analysis. This would also help to see how robust the results are, which parameters are more important and which parameters are less relevant, and to shed light on the key mechanisms.

(3) It is not clear what the main functional advantage of the specific E-I network model is compared to random networks. In terms of activity, they show that specific E-I networks amplify the input more than random networks (Fig. 3). But when it comes to classification, the effect seems to be very small (Fig. 5c). Description of different geometry of representation and manifold localization in specific networks compared to random networks is good, but it is more of an illustration of different activity patterns than proving a functional benefit for the network. The reader is still left with the question of what major functional benefits (in terms of computational/biological processing) should be expected from these networks, if they are to be a good model for olfactory processing and learning.<br /> One possibility for instance might be that the tasks used here are too easy to reveal the main benefits of the specific models - and more complex tasks would be needed to assess the functional enhancement (e.g. more noisy conditions or more combination of odours). It would be good to show this more clearly - or at least discuss it in relation to computation and function.

Reviewer #1 (Public Review):

Assessment:

This important work advances our understanding of navigation and path integration in mammals by using a clever behavioral paradigm. The paper provides compelling evidence that mice are able to create and use a cognitive map to find "short cuts" in an environment, using only the location of rewards relative to the point of entry to the environment and path integration, and need not rely on visual landmarks.

Summary:

The authors have designed a novel experimental apparatus called the 'Hidden Food Maze (HFM)' and a beautiful suite of behavioral experiments using this apparatus to investigate the interplay between allothetic and idiothetic cues in navigation. The results presented provide a clear demonstration of the central claim of the paper, namely that mice only need a fixed start location and path integration to develop a cognitive map. The experiments and analyses conducted to test the main claim of the paper -- that the animals have formed a cognitive map -- are conclusive. While I think the results are quite interesting and sound, one issue that needs to be addressed is the framing of how landmarks are used (or not), as discussed below, although I believe this will be a straightforward issue for the authors to address.

Strengths:

The 90-degree rotationally symmetric design and use of 4 distal landmarks and 4 quadrants with their corresponding rotationally equivalent locations (REL) lends itself to teasing apart the influence of path integration and landmark-based navigation in a clever way. The authors use a really complete set of experiments and associated controls to show that mice can use a start location and path integration to develop a cognitive map and generate shortcut routes to new locations.

Weaknesses:

I have two comments. The second comment is perhaps major and would require rephrasing multiple sentences/paragraphs throughout the paper.

(1) The data clearly indicate that in the hidden food maze (HFM) task mice did not use external visual "cue cards" to navigate, as this is clearly shown in the errors mice make when they start trials from a different start location when trained in the static entrance condition. The absence of visual landmark-guided behavior is indeed surprising, given the previous literature showing the use of distal landmarks to navigate and neural correlates of visual landmarks in hippocampal formation. While the authors briefly mention that the mice might not be using distal landmarks because of their pretraining procedure - I think it is worth highlighting this point (about the importance of landmark stability and citing relevant papers) and elaborating on it in greater detail. It is very likely that mice do not use the distal visual landmarks in this task because the pretraining of animals leads to them not identifying them as stable landmarks. For example, if they thought that each time they were introduced to the arena, it was "through the same door", then the landmarks would appear to be in arbitrary locations compared to the last time. In the same way, we as humans wouldn't use clouds or the location of people or other animate objects as trusted navigational beacons. In addition, the animals are introduced to the environment without any extra-maze landmarks that could help them resolve this ambiguity. Previous work (and what we see in our dome experiments) has shown that in environments with 'unreliable' landmarks, place cells are not controlled by landmarks - https://www.sciencedirect.com/science/article/pii/S0028390898000537, https://pubmed.ncbi.nlm.nih.gov/7891125/. This makes it likely that the absence of these distal visual landmarks when the animal first entered the maze ensured that the animal does not 'trust' these visual features as landmarks.

(2) I don't agree with the statement that 'Exogenous cues are not required for learning the food location'. There are many cues that the animal is likely using to help reduce errors in path integration. For example, the start location of the rat could act as a landmark/exogenous cue in the sense of partially correcting path integration errors. The maze has four identical entrances (90-degree rotationally symmetric). Despite this, it is entirely plausible that the animal can correct path integration errors by identifying the correct start entrance for a given trial, and indeed the distance/bearing to the others would also help triangulate one's location. Further, the overall arena geometry could help reduce PI error. For example, with a food source learned to be "near the middle" of the arena, the animal would surely not estimate the position to be near the far wall (and an interesting follow-on experiment would be to have two different-sized, but otherwise nearly identical arenas). As the rat travels away from the start location, small path integration errors are bound to accumulate, these errors could be at least partially corrected based on entrance and distal wall locations. If this process of periodically checking the location of the entrance to correct path integration errors is done every few seconds, path integration would be aided 'exogenously' to build a cognitive map. While the original claim of the paper still stands, i.e. mice can learn the location of a hidden food size when their starting point in the environment remains constant across trials. I would advise rewording portions of the paper, including the discussion throughout the paper that states claims such as "Exogenous cues are not required for learning the food location" to account for the possibility that the start and the overall arena geometry could be used as helpful exogenous cues to correct for path integration errors.

Reviewer #1 (Public Review):

The paper submitted by Yogesh and Keller explores the role of cholinergic input from the basal forebrain (BF) in the mouse primary visual cortex (V1). The study aims to understand the signals conveyed by BF cholinergic axons in the visual cortex, their impact on neurons in different cortical layers, and their computational significance in cortical visual processing. The authors employed two-photon calcium imaging to directly monitor cholinergic input from BF axons expressing GCaMP6 in mice running through a virtual corridor, revealing a strong correlation between BF axonal activity and locomotion. This persistent activation during locomotion suggests that BF input provides a binary locomotion state signal. To elucidate the impact of cholinergic input on cortical activity, the authors conducted optogenetic and chemogenetic manipulations, with a specific focus on L2/3 and L5 neurons. They found that cholinergic input modulates the responses of L5 neurons to visual stimuli and visuomotor mismatch, while not significantly affecting L2/3 neurons. Moreover, the study demonstrates that BF cholinergic input leads to decorrelation in the activity patterns of L2/3 and L5 neurons.

This topic has garnered significant attention in the field, drawing the interest of many researchers actively investigating the role of BF cholinergic input in cortical activity and sensory processing. The experiments and analyses were thoughtfully designed and conducted with rigorous standards, providing evidence of layer-specific differences in the impact of cholinergic input on neuronal responses to bottom-up (visual stimuli) and top-down inputs (visuomotor mismatch).

Reviewer #1 (Public Review):

The impact of this paper is that it shows conclusively the bone defects caused by ninein depletion, albeit transient defects, which has been indirectly deduced in past studies. The paper is largely descriptive including the cytoskeletal analysis of osteoclasts thus it remains unclear how ninein reduction causes bone defects and why this defect is transient. The Discussion includes several unfounded potential mechanisms that really need to be thoroughly analyzed to gain a mechanistic understanding of the bone defects in ninein-null mice.

Other points:<br /> Data showing normal osteoblasts in ninein-null mice was qualitative and requires further in-depth analysis and quantification of osteoblast and osteocyte presence and activity in ninein del/del mice to strengthen the study.

In ninein knock-out mice, reduced TRAP+ve multinuclear cells were observed (Figure 6A and 6B). However, the magnitude of difference (about 5% decrease in multinucleated cells) is not consistent with the skeletal deformities reported in Figures 2-4, potentially suggesting the contribution of additional mechanisms.

The fusion assay in Figure 6C needs further clarification. How was the syncytia perimeter defined to measure cell surface? The x-axis suggests that there are syncytia that contain up to 160 nuclei at day 3. How were the nuclei differentially stained and quantified?

Some text needs clarification. For instance, "On days 3 and 4, we found only about half as many large syncytia in cultures from ninein-deleted mice, compared to controls, but on day 5 large syncytia lacking ninein exceeded 90% of control levels. Altogether, this suggests that fusion deficiencies are a transient phenomenon in in vitro-induced adult osteoclasts. On later days of culture, fusion efficiency started to diminish." What is the definition of "large syncytia"? Is the fusion index increase by day 5 diminished in later days? A graph of the syncytia size/ nuclei number or fusion index in the above-mentioned days will be helpful.

Assessment of resorption was qualitative in Figure 6E and since the fusion deficiencies are transient, quantification of a corresponding resorption activity is needed. This should be described in the Materials and Methods section.

Further experiments are needed to show connections between reduced centrosome clustering and reduced osteoclast formation as there is no evidence to date that suggest centrosome clustering is required for cell fusion. Multi-color live imaging and dynamic analysis can be used to determine if the ninein deficient cells show defective movement/migration/ fusion dynamics.

Quantification of the % of multinucleated osteoclasts that contain clustered and dispersed centrosomes is needed.

Reviewer #1 (Public Review):

Summary:

Previous work in humans and non-human animals suggests that during offline periods following learning, the brain replays newly acquired information in a sequential manner. The present study uses a MEG-based decoding approach to investigate the nature of replay/reactivation during a cued recall task directly following a learning session, where human participants are trained on a new sequence of 10 visual images embedded in a graph structure. During retrieval, participants are then cued with two items from the learned sequence, and neural evidence is obtained for the simultaneous or sequential reactivation of future sequence items. The authors find evidence for both sequential and clustered (i.e., simultaneous) reactivation. Replicating previous work, low-performing participants tend to show sequential, temporally segregated reactivation of future items, whereas high-performing participants show more clustered reactivation. Adding to previous work, the authors show that an image's reactivation strength varies depending on its proximity to the retrieval cue within the graph structure.

Strengths:

As the authors point out, work on memory reactivation has largely been limited to the retrieval of single associations. Given the sequential nature of our real-life experiences, there is clearly value in extending this work to structured, sequential information. State-of-the-art decoding approaches for MEG are used to characterize the strength and timing of item reactivation. The manuscript is very well written with helpful and informative figures in the main sections. The task includes an extensive localizer with 50 repetitions per image, allowing for stable training of the decoders and the inclusion of several sanity checks demonstrating that on-screen items can be decoded with high accuracy.

Weaknesses:

Of major concern, the experiment is not optimally designed for analysis of the retrieval task phase, where only 4 min of recording time and a single presentation of each cue item are available for the analyses of sequential and non-sequential reactivation. In their revision, the authors include data from the learning blocks in their analysis. These blocks follow the same trial structure as the retrieval task, and apart from adding more data points could also reveal a possible shift from sequential to clustered reactivation as learning of the graph structure progresses. The new analyses are not entirely conclusive, maybe given the variability in the number of learning blocks that participants require to reach the criterion. In principle, they suggest that reactivation strength increases from learning (pre-rest) to final retrieval (post-rest).

On a more conceptual note, the main narrative of the manuscript implies that sequential and clustered reactivation are mutually exclusive, such that a single participant would show either one or the other type. With the analytic methods used here, however, it seems possible to observe both types of reactivation. For example, the observation that mean reactivation strength (across the entire trial, or in a given time window of interest) varies with graph distance does not exclude the possibility that this reactivation is also sequential. In fact, the approach of defining one peak time window of reactivation may bias towards simultaneous, graded reactivation. It would be helpful if the authors could clarify this conceptual point. A strong claim that the two types of reactivation are mutually exclusive would need to be substantiated by further evidence, for instance, a suitable metric contrasting "sequenceness" vs "clusteredness".

On the same point, the non-sequential reactivation analyses use a time window of peak decodability that is determined based on the average reactivation of all future items, irrespective of graph distance. In a sequential forward cascade of reactivations, it could be assumed that the reactivation of near items would peak earlier than the reactivation of far items. In the revised manuscript, the authors now show the "raw" timecourses of item decodability at different graph distances, clearly demonstrating their peak reactivation times, which show convincingly that reactivation for near and far items occurs at very similar time points. The question that remains, therefore, is whether the method of pre-selecting a time window of interest described above could exert a bias towards finding clustered reactivation.

Reviewer #1 (Public Review):

Summary:

Chang et al. provide glutamate co-expression profiles in the central noradrenergic system and test the requirement of Vglut2-based glutamatergic release in respiratory and metabolic activity under physiologically relevant gas challenges. Their experiments show that conditional deletion of Vglut2 in NA neurons does not impact steady-state breathing or metabolic activity in room air, hypercapnia, or hypoxia. Their observations challenge the importance of glutamatergic signaling from Vglut2 expressing NA neurons in normal respiratory homeostasis in conscious adult mice.

Strengths:

The comprehensive Vglut1, Vglut2, and Vglut3 co-expression profiles in the central noradrenergic system and the combined measurements of breathing and oxygen consumption are two major strengths of this study. Observations from these experiments provide previously undescribed insights into (1) expression patterns for subtypes of the vesicular glutamate transporter protein in the noradrenergic system and (2) the dispensable nature of Vglut2-dependent glutamate signaling from noradrenergic neurons to breathing responses to physiologically relevant gas challenges in adult conscious mice.

Weaknesses:

Although the cellular expression profiles for the vesicular glutamate transporters are provided, the study does not document that glutamatergic-based signaling originating from noradrenergic neurons is evident at the cellular level under normal, hypoxic, and/or hypercapnic conditions. The authors effectively recognize this issue and appropriately discuss their findings in this context.

Reviewer #1 (Public Review):

Summary:

The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

Strengths and weaknesses:

(1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. Why is no eGFP readout given in Figure 1C as for WT HSV? The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); so the story seems incomplete.

(2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.

(3) The primate groups are too small and the results to variable to make averages. In Figure 5, the group with ART and saline has two slow rebounders. It is not correct to average those with a single quick rebounder. Here the interpretation is NOT supported by the data.

Discussion

HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

Reviewer #1 (Public Review):

Summary:

The authors study age-related changes in the excitability and firing properties of sympathetic neurons, which they ascribe to age-related changes in the expression of KCNQ (Kv7, "M-type") K+ currents in rodent sympathetic neurons, whose regulation by GPCRs has been most thoroughly studied for over 40 years.

Strengths:

The strengths include the rigor of the current-clamp and voltage-clamp experiments and the lovely, crisp presentation of the data, The separation of neurons into tonic, phasic and adapting classes is also interesting, and informative. The ability to successfully isolate and dissociate peripheral ganglia from such older animals is also quite rare and commendable! There is much useful detail here.

Weaknesses:

Whereas the description of the data are very nice and useful, the manuscript does not provide much in the way of mechanistic insights. As such, the effect is more of an epi-phenomenon of unclear insight, and the authors cannot ascribe changes in signaling mechanisms, such as that of M1 mAChRs to the phenomena that is supported by data.

Joint Public Review:

Summary:

The paper explores chemosensory behaviour in surface and cave morphs and F2 hybrids in the Mexican cave fish Astyanax mexicanus. The authors develop a new behavioural assay for the long-term imaging of individual fish in a parallel high-throughput setup. The authors first demonstrate that the different morphs show different basal exploratory swimming patterns and that these patterns are stable for individual fish. Next, the authors test the attraction of fish to various concentrations of alanine and other amino acids. They find that the cave morph is a lot more sensitive to chemicals and shows directional chemotaxis along a diffusion gradient of amino acids. Surface fish, although can detect the chemicals, do not show marked chemotaxis behaviour and have an overall lower sensitivity. These differences have been reported previously but the authors report longer-term observations on many individual fish of both morphs and their F2 hybrids. The data also indicate that the observed behaviour is a quantitative genetic trait. The approach presented will allow the mapping of genes contribution to these traits. The work will be of general interest to behavioural neuroscientists and those interested in olfactory behaviours and the individual variability in behavioural patterns.

Strengths:

The authors provide a large dataset of swimming behaviour for surface fish and cave fish and also their F2 hybrids, demonstrating large differences in chemosensory behaviour and indicating that this is a quantitative genetic trait.

One strength of the paper is the development of a new and improved setup for the behavioural imaging of individual fish for extended periods and under chemosensory stimulation. The authors show that cave fish need up to 24 h of habituation to display a behavioural pattern that is consistent and unlikely to be due to the stressed state of the animals. The setup also uses relatively large tanks that allows the build-up of chemical gradients.

With their new system, the authors could generate cleaner results without mechanical disturbances. The authors characterize multiple measurements to score the odour response behaviours and also developed a new personality analysis. Their conclusion that cave fish evolved as a specialist to sense alanine and histidine among 6 tested amino acids was well supported by their data.

Weaknesses:

Further work will be needed to pinpoint the nature of the genetic changes and neurobiological mechanisms that underlie the differences between the two forms and the large individual variation of behaviours.<br /> The authors did not measure the concentrations of alanine and other amino acids in the local cave water and surface water.

Reviewer #1 (Public Review):

Summary:

The manuscript aims to provide insights into the mediators and mechanisms underlying tardigrade radiation tolerance. The authors start by assessing the effect of ionizing radiation (IR) on the tardigrade lab species, H. exemplaris, as well as the ability of this organism to recover from this stress - specifically they look at DNA double and single strand breaks. They go on to characterize the response of H. exemplaris and two other tardigrade species to IR at the transcriptomic level. Excitingly, the authors identify a novel gene/protein called TDR1 (tardigrade DNA damage response protein 1). They carefully assess the induction of expression/enrichment of this gene/protein using a combination of transcriptomics and biochemistry - even going so far as to use a translational inhibitor to confirm the de novo production of this protein. TDR1 binds DNA in vitro and co-localizes with DNA in tardigrades.

Reverse genetics in tardigrades is difficult, thus the authors use a heterologous system (human cells) to express TDR1 in. They find that when transiently expressed TDR1 helps improve human cell resistance to IR.

This work is a masterclass in integrative biology incorporating a holistic set of approaches spanning next-gen sequencing, organismal biology, biochemistry, and cell biology. I think the importance of the findings is suitable and honestly, I find very little to critique in their experimental approaches.

Overall, I find this to be one of the more compelling papers on tardigrade stress-tolerance I have read.

Reviewer #1 (Public Review):

Summary:

This work by Leclercq and colleagues performed metabolomics on biospecimens collected from 96 patients diagnosed with several types of alcohol use disorders (AUD). The authors discovered strong alterations in circulating glycerophospholipids, bile acids, and some gut microbe-derived metabolites in AUD patients compared to controls. An exciting part of this work is that metabolomics was also performed in frontal cortex of post-mortem brains and cerebrospinal fluid of heavy alcohol users, and some of the same metabolites were seen to be altered in the central nervous system. This is an important study that will form the basis for hypothesis generation around diet-microbe-host interactions in alcohol use disorder. The work is done in a highly rigorous manner, and the rigorously collected human samples are a clear strength of this work. Overall, many new insights may be gained by this work, and it is poised to have a high impact on the field.

Strengths:

(1) The rigorously collected patient-derived samples.

(2) There is high rigor in the metabolomics investigation.

(3) Statistical analyses are well-described and strong.

(4) An evident strength is the careful control of taking blood samples at the same time of the day to avoid alterations in meal- and circadian-related fluctuations in metabolites.

Weaknesses:

(1) Some validation in animal models of ethanol exposure compared to pair-fed controls would help strengthen causal relationships between metabolites and alterations in the CNS.

(2) The classification of "heavy alcohol users" based on autopsy reports may not be that accurate.

(3) The fact that most people with alcohol use disorder choose to drink over eating food, there needs to be some more discussion around how dietary intake (secondary to heavy drinking) most likely has a significant impact on the metabolome.

Reviewer #1 (Public Review):

Summary:

Arman Angaji and his team delved into the intricate world of tumor growth and evolution, utilizing a blend of computer simulations and real patient data from liver cancer.

Strengths:

Their analysis of how mutations and clones are distributed within tumors revealed an interesting finding: tumors don't just spread from their edges as previously believed. Instead, they expand both from within and the edges simultaneously, suggesting a unique growth mode. This mode naturally indicates that external forces may play a role in cancer cells dispersion within the tumor. Moreover, their research hints at an intriguing phenomenon - the high death rate of progenitor cells and extremely slow pace in growth in the initial phase of tumor expansion. Understanding this dynamic could significantly impact our comprehension of cancer development.

Weaknesses:

It's important to note, however, that this study relies on specific computer models, metrics derived from inferred clones, and a limited number of patient data. While the insights gained are promising, further investigation is essential to validate these findings. Nonetheless, this work opens up exciting avenues for comprehending the evolution of cancers.

Reviewer #1 (Public Review):

Summary:

This study explores the neural control of muscle by decomposing the firing activity of constituent motor units from the grid of surface electromyography (EMG) in the Tibialis (TA) Anterior and Vastus Lateralis (VL) during isometric contractions. The study involves extensive samples of motor units across the broadest range of voluntary contraction intensities up to 80% of MVC. The authors examine the rate coding of the population of motor units, which describes the instantaneous firing rate of each motor unit as a function of muscle force. This relationship is characterized by a natural logarithm function that delineates two distinct phases: an initial phase with a steep acceleration in firing rate, particularly pronounced in low-threshold motor units, and a subsequent modest linear increase in firing rate, more significant in high-threshold motor units.

Strengths:

The study makes a significant contribution to the field of neuromuscular physiology by providing a detailed analysis of motor unit behavior during muscle contractions in a few ways.

(1) The significance lies in its comprehensive framework of motor unit activity during isometric contractions in a broad range of intensities, providing insights into the non-linear relationship between the firing rate and the muscle force. The extensive sample of motor units across the pool confirms the observation in animal studies in which the spinal motoneuron exhibits a discharge consisting of distinct phases in response to synaptic currents, under the influence of persistent inward currents. As such, it is now reasonable to state the human motor units across the pool are also under the control of gain modulation via some neuromodulatory effects in addition to synaptic inputs arising from ionotropic effects.

(2) The firing scheme across the entire motoneuron pool revealed in this study reconciles the discrepancy in firing organization under debate; i.e., whether it is 'onion skin' like or not (Heckman and Enoka 2012). The onion skin like model states that the low threshold motor units discharge higher than high threshold motor units and have been held for a long time because the firing behaviors were examined in a partial range of contraction force range due to technical limitations. This reconciliation is crucial because it is fundamental to modelling the organization of motor unit recruitment and rate coding to achieve a desired force generation to advance our understanding of motor control.

(3) The extensive data collection with a novel blind source separation algorithm on the expanded number of channels of surface EMG signal provides a robust dataset that enhances the reliability and validity of findings, setting a new standard for empirical studies in the field.

Collectively, this study fills several knowledge gaps in the field and advances our understanding of the mechanism underlying the isometric force generation.

Weaknesses:

Although the findings and claims based on them are mostly well aligned, some accounts of the methods and claims need to be clarified.

(1) The authors examine the input-output function of a motor unit by constructing models, using force as an input and discharge rate as an output. It sounds circular, or the other way around to use the muscle force as an input variable, because the muscle force is the result of motor unit discharges, not the cause that elicits the discharges. More specifically, as a result of non-linear interactions of synchronous and/or asynchronous discharges of a population of a given motoneuron pool that give rise to transient increase/maintenance in twitch force, the gross muscle force is attained. I acknowledge that it is extremely challenging experimentally to measure synaptic currents impinging upon the spinal motoneurons in human subjects and the author has an assumption that the force could be used as a proxy of synaptic currents. However, it is necessary to explicitly provide the caveats and rationale behind that. Force could be used as the input variable for modelling.

2) The authors examine the firing organizations in TA and VL in this study without explicit purposes and rationale for choosing these muscles. The lack of accounts makes it hard for the readers to interpret the data presented, particularly in terms of comparing the results from the different muscles.

(3) In the methods, the author described the manual curation process after applying the blind source separation algorithm. For the readers to understand the whole process of decomposition and to secure rigor and robustness of the analyses, it would be necessary to provide details on what exact curation is performed with what criteria.

(4) In Figure 3, the early recruited units tend to become untraceable in the higher range of contraction. This is more pronounced in the muscle VL. This limitation would ambiguate the whole firing curve along the force axis and therefore limitation and the applicability in the different muscles needs to be discussed.

(5) It is unclear how commonly the notion "the long-held belief that rate coding is similar across motor units from the same pool" is held among the community without a reference. Different firing organizations have been modelled and discussed in the seminal paper by Fuglevand et al. (1993), and as far as I understand, the debate has not converged to a specific consensus. As such, any reference would be required to support the claim the notion is widely recognized.

(6) The authors claim that the firing behavior as a function of force is well characterized by a natural logarithmic function, which consists of initial steep acceleration followed by a modest increase in firing rate. Arguably the gain modulation in firing rate could be attributed to a neuromodulatory effect on the spinal motoneuron, which has been suggested by a number of animal studies. However, the complexity of the interactions between ionotropic and neuromodulatory inputs to motoneurons may require further elucidation to fully understand the mechanisms of neural control; it is possible to consider the differential acceleration among different threshold motor units as a differential combinatory effect of ionotropic and neuromodulatory inputs, but it is not trivially determined how differentially or systematically the inputs are organized. Likewise, the authors make an account for the difference in firing rate between TA and VL in terms of different amounts or balances of excitatory and inhibitory inputs to the motoneuron pool, but again this could be explained by other factors, such as a different extent of neuromodulatory effects. To determine the complexity of the interactions, further studies will be warranted.

(7) It is unclear with the account " ... the bandwidth of muscle force is < 10Hz during isometric contraction" in the manuscript alone, and therefore, it is difficult to understand the following claim. It appears very interesting and crucial for motor unit discharge and force generation and maintenance because it would pose a question of why the discharge rate of most motor units is higher than 10Hz, despite the bandwidth being so limited, but needs to be elaborated.

(References)

Heckman, C. J. & Enoka, R. M. Motor unit. Comprehensive Physiology 2, 2629-2682 (2012).

Fuglevand, A. J., Winter, D. A. & Patla, A. E. Models of recruitment and rate coding organization in motor-unit pools. J Neurophysiol 70, 2470-2488 (1993).

Reviewer #1 (Public Review):

Summary:

In this valuable study, the authors found that the macrolide drug rapamycin, which is an important pharmacological tool in the clinic and the research lab, is less specific than previously thought. They provide solid functional evidence that rapamycin activates TRPM8 and develop an NMR method to measure the specific binding of a ligand to a membrane protein.

Strengths:

The authors use a variety of complementary experimental techniques in several different systems, and their results support the conclusions drawn.

Weaknesses:

Controls are not shown in all cases, and a lack of unity across the figures makes the flow of the paper disjointed. The proposed location of the rapamycin binding pocket within the membrane means that molecular docking approaches designed for soluble proteins alone do not provide solid evidence for a rapamycin binding pocket location in TRPM8, but the authors are appropriately careful in stating that the model is consistent with their functional experiments.

Impact:

This work provides still more evidence for the polymodality of TRP channels, reminding both TRP channel researchers and those who use rapamycin in other contexts that the adjective "specific" is only meaningful in the context of what else has been explicitly tested.

Reviewer #1 (Public Review):

Summary:

Motivated by the existence of different behavioral strategies (e.g. model-based vs. model-free), and potentially different neural circuits that underlie them, Venditto et al. introduce a new approach for inferring which strategies animals are using from data. In particular, they extend the mixture of agents (MoA) framework to accommodate the possibility that the weighting among different strategies might change over time. These temporal dynamics are introduced via a hidden Markov model (HMM), i.e. with discrete state transitions. These state transition probabilities and initial state probabilities are fit simultaneously along with the MoA parameters, which include decay/learning rate and mixture weightings, using the EM algorithm. The authors test their model on data from Miller et al., 2017, 2022, arguing that this formulation leads to (1) better fits and (2) improved interpretability over their original model, which did not include the HMM portion. Lastly, they claim that certain aspects of OFC firing are modulated by the internal state as identified by the MoA-HMM.

Strengths:

The paper is very well written and easy to follow, especially for one with a significant modeling component. Furthermore, the authors do an excellent job explaining and then disentangling many threads that are often knotted together in discussions of animal behavior and RL: model-free vs. model-based choice, outcome vs. choice-focused, exploration vs. exploitation, bias, preservation. Each of these concepts is quantified by particular parameters of their models. Model recovery (Fig. 3) is mostly convincing and licenses their fits to animal behavior later (although see below). While the specific claims made about behavior and neural activity are not especially surprising (e.g. the animals begin a session, in which rare vs. common transitions are not yet known, in a more exploratory mode), the MoA-HMM framework seems broadly applicable to other tasks in the field and useful for the purpose of quantification here.

Weaknesses:

The authors sometimes seem to equivocate on to what extent they view their model as a neural (as opposed to merely behavioral) description. For example, they introduce their paper by citing work that views heterogeneity in strategy as the result of "relatively independent, separable circuits that are conceptualized as supporting distinct strategies, each potentially competing for control." The HMM, of course, also relates to internal states of the animal. Therefore, the reader might come away with the impression that the MoA-HMM is literally trying to model dynamic, competing controllers in the brain (e.g. basal ganglia vs. frontal cortex), as opposed to giving a descriptive account of their emergent behavior. If the former is really the intended interpretation, the authors should say more about how they think the weighting/arbitration mechanism between alternative strategies is implemented, and how it can be modulated over time. If not, they should make this clearer.

Second, while the authors demonstrate that model recovery recapitulates the weight dynamics and action values (Fig. 3), the actual parameters that are recovered are less precise (Fig. 3 Supplement 1). The authors should comment on how this might affect their later inferences from behavioral data. Furthermore, it would be better to quantify using the R^2 score between simulated and recovered, rather than the Pearson correlation (r), which doesn't enforce unity slope and zero intercept (i.e. the line that is plotted), and so will tend to exaggerate the strength of parameter recovery.

Finally, the authors are very aware of the difficulties associated with long-timescale (minutes) correlations with neural activity, including both satiety and electrode drift, so they do attempt to control for this using a third-order polynomial as a time regressor as well as interaction terms (Fig. 7 Supplement 1). However, on net there does not appear to be any significant difference between the permutation-corrected CPDs computed for states 2 and 3 across all neurons (Fig. 7D). This stands in contrast to the claim that "the modulation of the reward effect can also be seen between states 2 and 3 - state 2, on average, sees a higher modulation to reward that lasts significantly longer than modulation in state 3," which might be true for the neuron in Fig. 7C, but is never quantified. Thus, while I am convinced state modulation exists for model-based (MBr) outcome value (Fig. 7A-B), I'm not convinced that these more gradual shifts can be isolated by the MoA-HMM model, which is important to keep in mind for anyone looking to apply this model to their own data.

Reviewer #1 (Public Review):

The authors studied how hippocampal connectivity gradients across the lifespan, and how these relate to memory function and neurotransmitter distributions. They observed older age with less distinct transitions and observed an association between gradient de-differentiation and cognitive decline.

This is overall an innovative and interesting study to assess gradient alterations across the lifespan and its associations to cognition.

The paper is well-written, and the methods appear sound and thoughtful. There are several strengths, including the inclusion of two independent cohorts, the use of gradient mapping and alignment techniques, and an overall sound statistical and analysis framework. There are several areas for potential improvements in the paper, and these are listed below:

(1) The reported D1 associations appear a bit post-hoc in the current work and I was unclear why the authors specifically focussed on dopamine here, as other transmitter systems are similar present at the level of the hippocampus and implicated in aging.

Moreover, the authors may be aware that multiple PET tracers are somewhat challenged in the mesiotemporal region. Is this the case for the D1 receptor as well? The hippocampus is a small and complex structure, and PET more of a low res technique so one would want to highlight and discuss the limitations of the correlations with PET maps here and/or evaluate whether the analysis adds necessary findings to the study.

From my (perhaps somewhat biased) perspective, it might be valuable to instead or in addition look at measures of hippocampal microstructure and how these relate to the functional aging effects. This could be done, if available, using data from the same subjects (eg based on quantitative MRI contrasts and/or structural MRI) and/or using contextualization findings as implemented in eg hippomaps.readthedocs.io

(2) Can the authors clarify why they did not replicate based on cohorts that are more widely used in the community and open access, such as CamCAN and/or HCP-Aging? It might connect their results with other studies if an attempt was made to also show that findings persist in either of these repositories.

(3) The authors applied TSM and related these parameters to topographic changes in the gradients. I was wondering whether and how such an approach controls for autocorrelation present in both the PET map and gradients. Could the authors clarify?

(4) The TSM approach quantifies the gradients in terms of x/y/z direction in a cartesian coordinate system. Wouldn't a shape intrinsic coordinate system in the hippocampus also be interesting, and perhaps even be more efficient to look at here (see eg DeKraker 2022 eLife or Paquola et al 2020 eLife)?

Reviewer #1 (Public Review):

Summary:

The paper describes a program developed to identify PPI-hot spots using the free protein structure and compares it to FTMap and SPOTONE, two webservers that they consider as competitive approaches to the problem. On the positive side, I appreciate the effort in providing a new webserver that can be tested by the community but have two major concerns as follows.

(1) The comparison to the FTMap program is wrong. The authors misinterpret the article they refer to, i.e., Zerbe et al. "Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces" J. Chem. Inf. Model. 52, 2236-2244, (2012). FTMap identifies hot spots that bind small molecular ligands. The Zerbe et al. article shows that such hot spots tend to interact with hot spot residues on the partner protein in a protein-protein complex (emphasis on "partner"). Thus, the hot spots identified by FTMap are not the hot spots defined by the authors. In fact, because the Zerbe paper considers the partner protein in a complex, the results cannot be compared to the results of Chen et al. This difference is missed by the authors, and hence the comparison of the FTMap is invalid. I did not investigate the comparison to SPOTONE, and hence have no opinion.

(2) Chen et al. use a number of usual features in a variety of simple machine-learning methods to identify hot spot residues. This approach has been used in the literature for more than a decade. Although the authors say that they were able to find only FTMap and SPOTONE as servers, there are dozens of papers that describe such a methodology. Some examples are given here: (Higa and Tozzi, 2009; Keskin, et al., 2005; Lise, et al., 2011; Tuncbag, et al., 2009; Xia, et al., 2010). There are certainly more papers. Thus, while I consider the web server as a potentially useful contribution, the paper does not provide a fundamentally novel approach.

Higa, R.H. and Tozzi, C.L. Prediction of binding hot spot residues by using structural and evolutionary parameters. Genet Mol Biol 2009;32(3):626-633.

Keskin, O., Ma, B.Y. and Nussinov, R. Hot regions in protein-protein interactions: The organization and contribution of structurally conserved hot spot residues. J Mol Biol 2005;345(5):1281-1294.

Lise, S., et al. Predictions of Hot Spot Residues at Protein-Protein Interfaces Using Support Vector Machines. PLoS One 2011;6(2).

Tuncbag, N., Gursoy, A. and Keskin, O. Identification of computational hot spots in protein interfaces: combining solvent accessibility and inter-residue potentials improves the accuracy. Bioinformatics 2009;25(12):1513-1520.

Xia, J.F., et al. APIS: accurate prediction of hot spots in protein interfaces by combining protrusion index with solvent accessibility. BMC Bioinformatics 2010;11:174.

Strengths:<br /> A new web server was developed for detecting protein-protein interaction hot spots.

Weaknesses:<br /> The comparison to FTMap results is wrong. The method is not novel.

Reviewer #1 (Public Review):

Summary:

Balasubramanian et al. characterized the cell types comprising mouse Schlemm's canal (SC) using bulk and single-cell RNA sequencing (scRNA-seq). The results identify expression patterns that delineate the SC inner and outer wall cells and two inner wall 'states'. Further analysis demonstrates expression patterns of glaucoma-associated genes and receptor-ligand pairs between SEC's and neighboring trabecular meshwork.

Strengths:

While mouse SC has been profiled in previous scRNA-seq studies (van Zyl et al 2020, Thomson et al 2021), these data provide higher resolution of SC cell types, particularly endothelial cell (SEC) populations. SC is an important regulator of anterior chamber outflow and has important consequences for glaucoma.

Weaknesses:

(1) Since SC has previously been characterized in mouse, human, and other species by scRNA-seq in other studies, this study would benefit from more direct comparisons to published datasets. For example, Table 4 could be expanded to list the SC cell numbers profiled in each study. Expression patterns highlighted in this study could be independently verified by plotting in publicly available mouse SC datasets. Further, a comparison to human expression patterns would assess whether type-specific expression patterns are conserved. Alternatively, an integrated analysis could be performed. Indeed, the authors mention that an integrated analysis was attempted but the data is not shown. It is unclear if this was because of a lack of agreement between datasets or other reasons.

(2) Figure 1 presents bulk RNA seq results comparing SEC, BEC, and LEC expression patterns. These populations were isolated using cell surface markers and enrichment by FACS. Since each EC population is derived from the same sample, the accuracy of this data hinges on the purity of enrichment. However, a reference is not given for this method and it is not clear how purity was validated. The authors later note that marker Emcn, which was used to identify BECs, is also expressed in SECs and LECs at lower levels. It should be demonstrated that these populations are clearly separated by flow cytometry.

(3) Bulk RNA-seq analysis infers similarity from the number of DEGs between samples, however, this is not a robust indicator. A correlation analysis should be run to verify conclusions.

(4) Figures 2-4 present three different datasets targeting the same tissue: 1) C57bl/6j scRNA-seq, 2) C57bl/6j snRNA-seq, 3) 129/sj scRNA-seq. Integrated analysis comparing datasets #1 to #2 and #3 is also presented. Integration methods are not described beyond 'normalization for cell numbers'. It is unclear if additional alignment methods were used. Integration across each of these datasets needs careful consideration, especially since different filtering methods were used (e.g. <20% mito in scRNA-seq and <5% in snRNA-seq). Improper integration could affect the ability to cluster or exaggerate differences between cell/types and states. It would be useful to demonstrate the contribution of different samples and datasets to each cell type/state to verify that these are not driven by batch effects, mouse strain, or collection platform.

(5) IW1 and IW2 are not well separated, and it is unclear if these represent truly different cell states. Figure 5b shows the staining of CCL21A and describes expression in the 'posterior portion' but in the image there are no DAPI+ nuclei in the anterior portion, suggesting the sampling in this section is different from Figure 5a. This would be improved by co-staining NPNT and CCL21A to demonstrate specificity.

(6) The substructures observed within clusters in sc/snRNA-seq data suggest that overall profiling may still not be comprehensive. This should be noted in the discussion.

Reviewer #1 (Public Review):

Summary:

This study reports single-cell RNA sequencing results of lung adenocarcinoma, comparing 4 treatment-naive and 5 post-neoadjuvant chemotherapy tumor samples.<br /> The authors claim that there are metabolic reprogramming in tumor cells as well as stromal and immune cells after chemotherapy.<br /> The most significant findings are in the macrophages that there are more pro-tumorigenic cells after chemotherapy, i.e. CD45+CD11b+ARG+ cells. In the treatment-naive samples, more anti-tumorigenic CD45+CD11b+CD86+ macrophages are found. They sorted each population and performed functional analyses.

Strengths:

Comparison of the treatment-naive and post-chemotherapy samples of lung adenocarcinoma.

Weaknesses:

(1) Lengthy descriptive clustering analysis, with indistinct direct comparisons between the treatment-naive and the post-chemotherapy samples.<br /> (2) No statistical analysis was performed for the comparison.<br /> (3) Difficult to match data to the text.<br /> (4) ARG1 is a cytosolic enzyme that can be detected by intracellular staining after fixation. It is unclear how the staining and sorting was performed to measure function of sorted cells.

Reviewer #1 (Public Review):

In this manuscript, Molnar, Suranyi and colleagues have probed the genomic stability of Mycobacterium smegmatis in response to several anti-tuberculosis drugs as monotherapy and in combination. Unlike the study by Nyinoh and McFaddden http://dx.doi.org/10.1002/ddr.21497 (which should be cited), the authors use a sub-lethal dose of antibiotic. While this is motivated by sound technical considerations, the biological and therapeutic rationale could be further elaborated. The results the authors obtain are in line with papers examining the genomic mutation rate in vitro and from patient samples in Mycobacterium tuberculosis, in vitro in Mycobacterium smegmatis and in vitro in Mycobacterium tuberculosis (although the study by HL David (PMID: 4991927) is not cited). The results are confirmatory of previous studies. It is therefore puzzling why the authors propose the opposite hypothesis in the paper (i.e antibiotic exposure should increase mutation rates) merely to tear it down later. This straw-man style is entirely unnecessary. The results on the nucleotide pools are interesting, but the statistically significant data is difficult to identify as presented, and therefore the new biological insights are unclear. Finally, the authors show that a fluctuation assay generates mutations with higher frequencies that the genetic stability assays, confirming the well-known effect of phenotypic antibiotic resistance.

Reviewer #1 (Public Review):

Summary:

The authors of this study aim to use an optimization algorithm approach, based on the established Nelder-Mead method, to infer polymer models that best match input bulk Hi-C contact data. The procedure infers the best parameters of a generic polymer model that combines loop-extrusion (LE) dynamics and compartmentalization of chromatin types driven by weak biochemical affinities. Using this and DNA FISH, the authors investigate the chromatin structure of the MYC locus in leukemia cells, showing that loop extrusion alone cannot explain local pathogenic chromatin rearrangements. Finally, they study the locus single-cell heterogeneity and time dynamics.

Strengths:

-The optimization method provides a fast computational tool that speeds up the parameter search of complex chromatin polymer models and is a good technical advancement.

-The method is not restricted to short genomic regions, as in principle it can be applied genome-wide to any input Hi-C dataset, and could be potentially useful for testing predictions on chromatin structure.

Weaknesses:

(1) The optimization is based on the iterative comparison of simulated and Hi-C contact matrices using the Spearman correlation. However, the inferred set of the best-fit simulation parameters could sensitively depend on such a specific metric choice, questioning the robustness of the output polymer models. How do results change by using different correlation coefficients?

(2) The best-fit contact threshold of 420nm seems a quite large value, considering that contact probabilities of pairs of loci at the mega-base scale are defined within 150nm (see, e.g., Bintu et al. Science (2018) and Takei et al. Science (2021)).

(3) In their model, the authors consider the presence of LE anchor sites at Hi-C TAD boundaries. Do they correspond to real, experimentally found CTCF sites located at genomic positions, or they are just assumed? A track of CTCF peaks of the considered chromatin loci would be needed.

(4) In the model, each TAD is assigned a specific energy affinity value. Do the different domain types (i.e., different colors) have a mutually attractive energy? If so, what is its value and how is it determined? The simulated contact maps (e.g., Figure 2C) seem to allow attractions between different blocks, yet this is unclear.

(5) To substantiate the claim that the simulations can predict heterogeneity across single cells, the authors should perform additional analyses. For instance, they could plot the histograms (models vs. experiments) of the TAD2-TAD4 distance distributions and check whether the models can recapitulate the FISH-observed variance or standard deviation. They could also add other testable predictions, e.g., on gyration radius distributions, kurtosis, all-against-all comparison of single-molecule distance matrices, etc,.

(6) The authors state that loop extrusion is crucial for enhancer function only at large distances. How does that reconcile, e.g., with Mach et al. Nature Gen. (2022) where LE is found to constrain the dynamics of genomically close (150kb) chromatin loci?

Reviewer #1 (Public Review):

Summary:

In the work: "Endosomal sorting protein SNX4 limits synaptic vesicle docking and release" Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly developed in vitro KO model, with live imaging experiments, electrophysiological recordings, and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with a higher number of docked vesicles and shorter AZ. The evaluation of the presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that resulted in chronical deletion of SNX4 in neurons. A convincing sequence of experimental techniques is applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at the presynaptic site is relevant and of high interest in the field of synaptic physiology and in the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow us to address the physiological roles of SNX4 at the synaptic site and speculate on the underlying molecular mechanisms. In addition, due to the described role of SNX4 in autophagy and the high interest in the regulation of synaptic autophagy in the field of synaptic physiology, an initial evaluation of the autophagy phenotype in the neuronal SNX4KO model is important, and not to be only restricted to the discussion section.

Reviewer #1 (Public Review):

In this study, the authors explore the implications of two types of rhythmic inhibition - "gamma" (30-80 Hz) and "beta"(13-30Hz) - for synaptic integration. They study this in a multi-compartmental model L5 pyramidal neuron with Poisson excitation and rhythmic inhibition (16 Hz and 64 Hz), applied either to the perisomatic or apical tuft regions in the neuron. They find that 64 Hz inhibition applied to the cell body is effective in phasic modulation of AP generation, while 16 Hz inhibition applied to the apical tufts is effective in phasic modulation of dendritic spikes (in addition to APs). Switching the location of the two kinds of rhythmic inhibition reduces the overall excitability, but is not effective in phasic modulation of either dendritic spikes and weakly so for somatic APs.

Strengths:

The effect of the timescale of rhythmic inhibition on synaptic integration is an interesting question, since a) rhythmic spiking is most strongly evident in inhibitory population, b) rhythmic spiking is modulated by behavioral states and the sensory environment. The methods are clear and the data are well-presented. The study systematically explores the effect of two frequencies of rhythmic inhibition in a biophysically detailed model. The study considers not only idealized rhythmic inhibition but also the bursty kind that is observed in in-vivo conditions. Both distributed and clustered excitatory synaptic organization are simulated, which covers the two extremes of the spatial organization of excitatory inputs in-vivo.

Weaknesses:

SOM+ interneurons such as Martinotti cells target the apical tufts of pyramidals in the cortex. Since interneurons in general are strongly implicated in mediating rhythmic population activity over a range of timescales, it is quite appropriate to study the consequence of rhythmic inhibition provided by SOM+ interneurons for synaptic integration, including the phenomenon of dendritic spikes. However, using conclusions from a singular study (ref 22) to identify the beta band as the rhythm mediated by SOM+ is not very accurate. SOM+ interneurons have been implicated in regulating rhythms centered just below 30 Hz (refs 22, 21). It is a range that lies in the grey zone of the traditional definition of beta and gamma. However, it is significantly higher than the 16 Hz rhythms explored in this study. It thus remains unknown how a 25-30 Hz rhythmic inhibition (that has an experimentally suggested role for dendrite targeting SOM+ INs) in apical tufts regulates dendritic spikes.

Distal dendritic inhibition has been previously shown to be more effective in controlling dendritic spikes. However, given the slow timescale of dendritic spikes, it can be hypothesized that high-frequency rhythmic inhibition would be ineffective in entraining the dendritic spikes either in distal or proximal location, as demonstrated by 4H and 5F, and vice versa. A computational study can take this further by exploring the robustness of this hypothesis. By sticking to a single-frequency definition of what constitutes Gamma (64 Hz) and Beta (16 Hz) inhibition, the current exploration does support the core hypothesis. However, given the temporal dynamics of dendritic spikes, it is valuable to learn, for example, the upper bound of "Beta" range (13-30Hz) inhibition that fails to phasically modulate them. In addition to the reason stated in the earlier paragraph, Alpha band activity (8-12 Hz), has been implicated (e.g. van Kerkoerle, 2014) in signaling of inter-areal feedback to the superficial layer in the cortex, potentially targeting apical tufts of pyramidals from multiple layers and resulting in alpha-range rhythmic inhibition. To make the findings significant, it might therefore be more pertinent to understand the consequences of ~10Hz rhythmic inhibition (in addition to the ~25-30 Hz Beta/Gamma) in the apical tufts for phasic modulation of dendritic spikes.

The differential effect of Gamma and Beta range inhibition on basal and apical excitatory clusters is not convincing from the information provided. The basal cluster appears to overlap with perisomatic inhibitory synapses. The description in the methods does not have enough information to negate the visual perception (ln 979-81). With this understanding, it is not surprising that the correlation between excitation and APs is high (during the trough of gamma) for basal and not apical excitation. A more comparable scenario would be a more distal location of the basal excitatory cluster.

Reviewer #1 (Public Review):

Summary:

In this paper, Kalidini and Crevecoeur ask why sequential movements are sometimes coarticulated. To answer this question, first, they modified a standard optimal controller to perform consecutive reaches to two targets (T1 and T2). They investigated the optimal solution with and without a constraint on the endpoint's velocity in the via target (T1). They observed that the controller coarticulates the movements only when there is no constraint on the speed at the via-point. They characterized coarticulation in two ways: First, T2 affected the curvature of the first reach in unperturbed reaches. Second, T2 affected corrective movements in response to a mechanical perturbation of the first reach.

Parallel to the modeling work, they ran the same experiment on human participants. The participants were instructed to either consider T1 as via point (go task) or to slow down in T1 and then continue to T2 (stop task). Mirroring the simulation results, they observed coarticulation only in the go task. Interestingly, in the go task, when the initial reach was occasionally perturbed, the long-latency feedback responses differed for different T2 targets, suggesting that the information about the final target was already present in the motor circuits that mediate the long-latency response. In summary, they conclude that coarticulation in sequential tasks depends on instruction, and when coarticulation happens, the corrections in earlier segments of movement reflect the entirety of the coarticulated sequence.

Evaluation

Among many strengths of this paper, most notably, the results and the experiment design are grounded in, and guided by the optimal control simulation. The methods and procedures are appropriate and standard. The results and methods are explained sufficiently and the paper is written clearly. The results on modulation of long-latency response based on future goals are interesting and of broad interest for future experiments on motor control in sequential movement. However, I find the authors' framing of these results, mostly in the introduction section, somewhat complicated.

The current version of the introduction motivates the study by suggesting that "coarticulation and separation of sub-movement [in sequential movements] have been formulated as distinct hypotheses" and this apparent distinction, which led to contradictory results, can be resolved by Optimal Feedback Control (OFC) framework in which task-optimized control gains control coarticulation. This framing seems complicated for two main reasons. First, the authors use chunking and coarticulation interchangeably. However, as originally proposed by (Miller 1956), the chunking of the sequence items may fully occur at an abstract level like working memory, with no motoric coarticulation of sequence elements at the level of motor execution. In this scenario, sequence production will be faster due to the proactive preparation of sequence elements. This simple dissociation between chunking and coarticulation may already explain the apparent contradiction between the previous works mentioned in the introduction section. Second, the authors propose the OFC as a novel approach for studying neural correlates of sequence production. While I agree that OFC simulations can be highly insightful as a normative model for understanding the importance of sequence elements, it is unclear to me how OFCs can generate new hypotheses regarding the neural implementation of sequential movements. For instance, if the control gains are summarizing the instruction of the task and the relevance of future targets, it is unclear in which brain areas, or how these control gains are implemented. I believe the manuscript will benefit from making points more clear in the introduction and the discussion sections.

Reviewer #1 (Public Review):

Summary:

This study aimed at gaining a better comprehension of the functional role of acetylcholine release within the sensory cortex. To this end, the authors measured the dynamics of cortical acetylcholine release using two-photon imaging of the GRAB-Ach3.0 fluorescent sensor, either in the mouse primary somatosensory cortex (S1), throughout the learning of a whisker-dependent object position discrimination task, or in the primary auditory cortex (A1) of mice engaged in a specific sound signal detection task.

The illustrated results suggest that variations in acetylcholine release tend to be associated, in the primary sensory areas, with goal-directed actions (whisking in the case of the object position discrimination task, and more strongly with licking), rather than with sensory inputs or rewards. They also indicate that the variations in cholinergic signal specifically associated with licking increase with learning.

Strengths:

The impact of cholinergic inputs on cortical function has intrigued neuroscientists for many decades due to the complexity of its mode of action on the molecular and cellular points of view.

Being able to image the dynamics of cortical cholinergic release in vivo on mice engaged in goal-directed tasks has moved this field into a really exciting phase, where it becomes possible to draw links between specific behavioral features and local variations of cholinergic release in given cortical areas.

This study is therefore particularly timely, it provides a set of precious and original data. Globally the experiments were rigorously designed, and the illustrated quantifications and analyses follow high standards. This work therefore constitutes a valuable contribution to this field of research and could be of interest to a large audience.

Weaknesses:

Although the manuscript reports very interesting links between behavior and cortical cholinergic release, the study remains correlative and is devoid of experiments allowing to link causally cholinergic cortical inputs with motor actions, and more globally to gauge their impact on learning and execution of the tasks. Since the nature of the link between goal-directed motor actions and acetylcholine dynamics is not really clarified here, the word "drive" in the title of the paper, which may have a causal connotation should be replaced (especially since acetylcholine-related signal fluctuations seems often to precede motor actions).

As high-speed videography of the C2 whisker was achieved during the object position discrimination task, it seems that the whisker curvature changes could have been quantified in addition to the whisker angle. This would allow appreciation of how acetylcholine related signals vary according to both whisker-related motor output and sensory input, hereby providing clearer support for the assertion that acetylcholine levels are "related to motor actions rather than sensory inputs".

The data set related to the auditory task is used here to support the claim that licks rather than rewards are linked to variations of fluorescence of the cholinergic sensor in sensory cortices. These data seem very interesting indeed but are shown here in a very incomplete manner (a figure illustrating the learning curves of the 6 recorded animals, and acetylcholine dynamics during the four types of trials would be very welcome). If the animals were placed on a treadmill and the locomotion measured, together with pupil size, during the task as in Gee et al., BioRxiv 2022, one could ask how these other motor activities are linked with acetylcholine dynamics in A1. By comparing the impact of goal-directed actions versus motor activities accompanying more global state transitions on acetylcholine dynamics, these data could provide a particularly valuable contribution to this study. They could in addition rule out potential confounding factors regarding the claim that cholinergic dynamics are here mainly linked to first licks.

Coming back to the whisker-dependent object localization task, if cholinergic-related signals have been recorded during the "no whisker sessions", analyzing these data would be very useful in the scope of this study. Indeed, during these sessions, the animals were not naive, since they went through the learning of the task, but could not resolve it anymore, still they most probably kept on licking upon the pole-in and/or pole-out cues. In these sessions, the licking is fully dissociated from tactile sensory inputs, and for this reason it would be particularly interesting to see how the fluorescence varies with first licks. In addition, plotting these sessions in Figure 6C would be informative. Indeed, if the increase of cholinergic signals with performance comes progressively due to changes in the internal state of the animal and/or plasticity mechanisms, first lick related cholinergic signal variations could remain high despite the decrease of performance in these sessions.

Finally, because the functional role of cortical cholinergic release is a hot topic, a few recent studies addressing this question with slightly different approaches in the visual cortex would be worth mentioning, at least in the discussion, as well as a recent study focusing on motor learning, which revealed an apparent decrease of acetylcholine dynamics associated with goal-directed motor actions upon learning.

Reviewer #1 (Public Review):

Summary:

In this work, Wang and colleagues used Drosophila-Serratia as a host-microbe model to investigate the impact of the host on gut bacteria. The authors showed that Drosophila larvae reduce S. marcescens abundance in the food likely due to a combination of mechanical force and secretion of antimicrobial peptides. S. marcescens exposed to Drosophila larvae lost virulence to flies and could promote larval growth similar to typical Drosophila gut commensals. These phenotypic changes were reflected in the transcriptome and metabolome of bacteria, suggesting that the host could drive the switch from pathogenicity to commensalism in bacteria. Further, the authors used single-cell bacterial RNA-seq to demonstrate the heterogeneity in gut bacterial populations.

Strengths:

This is a valuable work that addresses an important question of the effect of the host on its gut microbes. The authors could convincingly demonstrate that gut bacteria are strongly affected by the host with important consequences for both interacting partners. Moreover, the authors used state-of-the-art bacterial single-cell RNA-seq to reveal heterogeneity in host-associated commensal populations.

Weaknesses:

Some of the conclusions are not fully supported by the data.

Specifically, in lines 142-143, the authors claim that larva antagonizes the pathogenicity of S. marcescens based on the survival data. I do not fully agree with this statement. An alternative possibility could be that, since there are fewer S. marcescens in larvae-processed food, flies receive a lower pathogen load and consequently survive. Can the authors rule this out?

Also, the authors propose that Drosophila larvae induce a transition from pathogenicity to commensalism in S. marcescens and provide nice phenotypic and transcriptomic data supporting this claim. However, is it driven only by transcriptional changes? Considering high mutation rates in bacteria, it is possible that S. marcescens during growth in the presence of larvae acquired mutations causing all the observed phenotypic and transcriptional changes. To test this possibility, the authors could check how long S. marcescens maintains the traits it acquires during growth with Drosophila. If these traits persist after reculturing isolated bacteria, it is very likely they are caused by genome alterations, if not - likely it is a phenotypic switch driven by transcriptional changes.

Reviewer #1 (Public Review):

Summary:

By using the biophysical chromosome stretching, the authors measured the stiffness of chromosomes of mouse oocytes in meiosis I (MI) and meiosis II (MII). This study was the follow-up of previous studies in spermatocytes (and oocytes) by the authors (Biggs et al. Commun. Biol. 2020: Hornick et al. J. Assist. Rep. and Genet. 2015). They showed that MI chromosomes are much stiffer (~10 fold) than mitotic chromosomes of mouse embryonic fibroblast (MEF) cells. MII chromosomes are also stiffer than the mitotic chromosomes. The authors also found that oocyte aging increases the stiffness of the chromosomes. Surprisingly, the stiffness of meiotic chromosomes is independent of meiotic chromosome components, Rec8, Stag3, and Rad21L. with aging.

Strengths:

This provides a new insight into the biophysical property of meiotic chromosomes, that is chromosome stiffness. The stiffness of chromosomes in meiosis prophase I is ~10-fold higher than that of mitotic chromosomes, which is independent of meiotic cohesin. The increased stiffness during oocyte aging is a novel finding.

Weaknesses:

A major weakness of this paper is that it does not provide any molecular mechanism underlying the difference between MI and MII chromosomes (and/or prophase I and mitotic chromosomes).

Reviewer #1 (Public Review):

Summary:<br /> This paper addresses the important question of the neural mechanisms underlying interval discrimination. The authors develop a detailed and biologically plausible model based on a previously proposed theory of timing. The model proposes that the interval between two stimuli can be encoded in the state of the neuronal and synaptic properties, specifically those with time constants on the order of hundreds of milliseconds, such as short-term synaptic plasticity and GABAb currents. Based on biological parameters in the PFC the authors show that the model can account for interval discrimination for up to 750 ms. Furthermore, the model accounts for three well-established psychophysical properties of interval timing: the linear relation between objective and neural time, the scalar property/Weber's law, and dopaminergic modulation of timing (although this property is less robust). Of particular novelty is the demonstration of Weber's law, and an explanation of how many complex and nonlinear neuronal properties produce a linear relationship between the standard deviation of interval estimates and their mean.

This is an interesting paper that addresses a significant gap in the field. However, I have one major concern. As I understood the methods (and I may have misunderstood) it seems that the readout units are not operating in continuous time, and that interval discrimination relies in part on external information. Specifically, the readout units only look at the spike counts during the window delta_t_w. Thus, discrimination between 100 and 200 ms looks only at the spikes at 120-145 and 220-245, respectively, meaning that the experimenters are providing interval information for the readout of the intervals being discriminated. If this is indeed the case the model is fairly limited in biological plausibility and significantly dampens my enthusiasm for the paper.

Stimulus onset occurs at 1500 ms in order to allow the network to stabilize. Ideally, this value should be randomized across trials to ensure performance generalizes across initial states.

Why does StDev saturate? Is that because subjective time saturates as well?

The model captures the effect of D2 receptors observed in some timing studies, specifically and DR2 activation increases "clock" speed. In the discussion, it would be nice to explain that dopaminergic modulation of subjective timing is not as universally observed as the linear psychophysical law or the scalar property, and I believe somewhat controversial (e.g., Ward, ..., Balsam, 2009).

(NB: Regarding my potential concern that that the decoding was performed in discontinuous time, the authors have clarified that decoding was done in continuous time--i.e., each output unit was trained to respond to a given time bin of the target interval but exposed to all time bins of all intervals during testing. Thus confirming the robustness of their decoding procedure and model.)

Reviewer #1 (Public Review):

The study by Prieto et al. faces the increasingly serious problem of bacterial resistance to antimicrobial agents. This work has an important element of novelty proposing a new approach to control antibiotic resistance spread by plasmids. Instead of targeting the resistance determinant, plasmid-borne proteins are used as antigens to be bound by specific nanobodies (Nbs). Once bound plasmid transfer was inhibited and Salmonella infection blocked. This in-depth study is quite detailed and complex, with many experiments (9 figures with multiple panels), rigorously carried out. Results fully support the authors' conclusions. Specifically, the authors investigated the role of two large molecular weight proteins (RSP and RSP2) encoded by the IncHI1 derivative-plasmid R27 of Salmonella. These proteins have bacterial Ig-like (Big) domains and are expressed on the cell surface, creating the opportunity for them to serve as immunostimulatory antigens. Using a mouse infection model, the authors showed that RSP proteins can properly function as antigens, in Salmonella strains harboring the IncHI1 plasmid. The authors clearly showed increased levels of specific IgG and IgA antibodies against these RSP proteins proteins in different tissues of immunized animals. In addition, non-immunized mice exhibited Salmonella colonization in the spleen and much more severe disease than immunized ones.

However, the strength of this work is the selection and production of nanobodies (Nbs) that specifically interact with the extracellular domain of RSP proteins. The procedure to obtain Nbs is lengthy and complicated and includes the immunization of dromedaries with purified RPS and the construction of a VHH (H-chain antibody variable region) library in E. coli. As RSP is expressed on the surface of E. coli, specific Nbs were able to agglutinate Salmonella strains harboring the p27 plasmid encoding the RSP proteins.<br /> The authors demonstrated that Nbs-RSP reduced the conjugation frequency of p27 thus limiting the diffusion of the amp resistance harbored by the plasmid. This represents an innovative and promising strategy to fight antibiotic resistance, as it is not blocked by the mechanism that determines, in the specific case, the amp resistance of p27 but it targets an antigen associated with HincHI- derivative plasmids. Thus, RPS vaccination could be effective not only against Salmonella but also against other enteric bacteria. A possible criticism could be that Nbs against RSP proteins reduce the severity of the disease but do not completely prevent the infection by Salmonella.

Reviewer #1 (Public Review):

Summary:

This is a nice paper taking a broad range of aspects and endpoints into account. The effect of GAHT in girls has been nicely worked out. Changes in Sertoli and peritubular cells appear valid, less strong evidence is provided for Leydig cell development. The recovery of SSCs appears an overjudgement and should be rephrased. The multitude and diversity of datasets appear a strength and a weakness as some datasets were not sufficiently critically reviewed and a selection of highlights provides a certain bias to the interpretation and conclusion of the study.

The authors need to indicate that the subset of data on SSCs has been reported previously (Human Reprod 36: 5-15 (2021) and is simply re-incorporated in the present paper. as Fig. 1C. There are sufficient new results to publish the remaining datasets as a separate paper. Authors could refer to the SSC data with reference to the previous publication.

Strengths:

The patient cohort is impressive and is nicely characterized. Here, histological endpoints and endocrine profiles were analyzed appropriately for most endpoints. The paper is well-written and has many new findings.

Weaknesses:

The patients and controls are poorly separated in regard to pubertal status. Here additional endpoints (e.g. Tanner status) would have been helpful especially as the individual patient history is unknown. Pre- and peri-puberty is a very rough differentiation. The characterization and evaluation of Leydig cells is the weakest histological endpoint. Here, additional markers may be required. Fig. 1 suffers from suboptimal micrograph quality.

Reviewer #1 (Public Review):

This manuscript describes the pattern of relaxed selection observed at spermatogenesis genes in gorillas, presumably due to the low sperm competition associated with single-male polygyny. The analyses to detect patterns of selection are very thorough, as are the follow up analyses to characterize the function of these genes. Furthermore, the authors take the extra steps of in vivo determination of function with a Drosophila model.

This is an excellent paper. It addresses the interesting phenomenon of relaxation of selection as a genomic signal of reproductive strategies using multiple computational approaches and follow-up analyses by pulling in data from GO, mouse knockouts, human infertility database, and even Drosophila RNAi experiments. I really appreciate the comprehensive and creative approach to analyze and explore the data. As far as I can tell, the analyses were performed soundly and statistics are appropriate. The Introduction and Discussion sections are thoughtful and well-written. I have no major criticisms of the manuscript.

The main area that I would suggest for improvement is in the "Caveats and Limitations" section of the Discussion. Currently, the first paragraph of this section states the obvious that genetic manipulation of gorillas is not feasible. Beyond a reminder to the reader that this was a rationale for the Drosophila work, it isn't really adding much insight. The second paragraph is a brief discussion of the directionality of change. I think it comes across as overly simplistic, with a sort of "well, we can never know" feel. Obviously, there are plenty of researchers who do model change to infer direction and causation, and there are plenty of published papers attempting to do so with respect to mating systems in primates.

I do not think the authors need to remove these paragraphs, but I do encourage them to turn the "Caveats and Limitations" section into something more meaningful by addressing limitations of the work that was actually done rather than limitations of hypothetical things that were not done. A few areas come to mind. First, the authors should discuss the effect of gene-tree vs species-tree inconsistencies in the analyses, which could affect the identification of gorilla-specific amino acid changes and/or the dN/dS estimates. Incomplete lineage sorting is very common in primates including the gorilla-chimp-human splits (Rivas-González et al. 2023). It would be nice to hear the authors' thoughts on how that might affect their analyses. Second, the dN/dS-based analyses assume the neutrality of synonymous substitutions. Of course, that assumption is not completely true; it might be true enough, and the authors should at least note it as a caveat. Third, and potentially related, is the consideration that these protein-coding genes may be functioning in other ways such as via antisense transcription. The genes under relaxed selection may be on their way to becoming pseudogenes and evolving as such at the sequence level, but many pseudogenes continue to be transcribed sense or anti-sense in a regulatory purpose. I don't think there is a way to incorporate this into the authors' analyses but it would be nice to see it acknowledged as a caveat or limitation.

Reviewer #1 (Public Review):

Summary:

In this report, Yu et al ascribe potential tumor suppressive functions to the non-core regions of RAG1/2 recombinases. Using a well-established BCR-ABL oncogene-driven system, the authors model the development of B cell acute lymphoblastic leukemia in mice and found that RAG mutants lacking non-core regions show accelerated leukemogenesis. They further report that the loss of non-core regions of RAG1/2 increases genomic instability, possibly caused by increased off-target recombination of aberrant RAG-induced breaks. The authors conclude that the non-core regions of RAG1 in particular not only increases the fidelity of VDJ recombination, but may also influence the recombination "range" of off-target joints, and that in the absence of the non-core regions, mutant RAG1/2 (termed cRAGs) catalyze high levels of off-target recombination leading to the development of aggressive leukemia.

Strengths:

The authors used a genetically defined oncogene-driven model to study the effect of RAG non-core regions have on leukemogenesis. The animal studies were well performed and generally included a good number of mice. Therefore, the finding that cRAG expression led to development of more aggressive BCR-ABL+ leukemia compared to fRAG is solid. The authors also present some nice analyses that characterize the (genomic) nature of aggressive leukemia that develop in the absence of RAG non-core regions.

Weaknesses:

The paper relies on cRAG1/2 overexpression, an experimental limitation that needs to be taken into consideration when extrapolating the physiological relevance of the findings.

Reviewer #1 (Public Review):

Summary:

This study explores the sequence characteristics and features of high-occupancy target (HOT) loci across the human genome. The computational analyses presented in this paper provide information into the correlation of TF binding and regulatory networks at HOT loci that were regarded as lacking sequence specificity.

By leveraging hundreds of ChIP-seq datasets from the ENCODE Project to delineate HOT loci in HepG2, K562, and H1-hESC cells, the investigators identified the regulatory significance and participation in 3D chromatin interactions of HOT loci. Subsequent exploration focused on the interaction of DNA-associated proteins (DAPs) with HOT loci using computational models. The models established that the potential formation of HOT loci is likely embedded in their DNA sequences and is significantly influenced by GC contents. Further inquiry exposed contrasting roles of HOT loci in housekeeping and tissue-specific functions spanning various cell types, with distinctions between embryonic and differentiated states, including instances of polymorphic variability. The authors conclude with a speculative model that HOT loci serve as anchors where phase-separated transcriptional condensates form. The findings presented here open avenues for future research, encouraging more exploration of the functional implications of HOT loci.

Strengths:

The concept of using computational models to define characteristics of HOT loci is refreshing and allows researchers to take a different approach to identifying potential targets. The major strengths of the study lies in the very large number of datasets analyzed, with hundreds of ChIP-seq data sets for both HepG2 and K562 cells as part of the ENCODE project. Such quantitative power allowed the authors to delve deeply into HOT loci, which were previously thought to be artifacts.

Weaknesses:

While this study contributes to our knowledge of HOT loci, there are critical weaknesses that need to be addressed. There are questions on the validity of the assumptions made for certain analyses. The speculative nature of the proposed model involving transcriptional condensates needs either further validation or be toned down. Furthermore, some apparent contradictions exist among the main conclusions, and these either need to be better explained or corrected. Lastly, several figure panels could be better explained or described in the figure legends.

Reviewer #1 (Public Review):

Summary:<br /> The study claims to explore plant microbiome engineering using host-mediated selection as a strategy to enhance rice growth and drought tolerance.

Strengths:

The authors have derived and identified simplified microbiomes from wild microbial communities of rice fields, deserts, and serpentine seep soils by selecting microbiomes from plants with desired phenotypes across generations. Metagenome-assembled genomes revealed enriched functions, such as glycerol-3-phosphate and iron transport, known to mediate plant-microbe interactions during drought.

Weaknesses:

The findings demonstrate the efficacy of host-mediated microbiome selection, but the engineering part for enhancing rice performance under drought-stress conditions has not been provided. The proposed mechanisms rely on correlations but not direct experimental proofs.

Reviewer #1 (Public Review):

Summary:

This short report shows that the transcription factor gene mirror is specifically expressed in the posterior region of the butterfly wing imaginal disk, and uses CRISPR mosaic knock-outs to show it is necessary to specify the morphological features (scales, veins, and surface) of this area.

Strengths:

The data and figures support the conclusions. The article is swiftly written and makes an interesting evolutionary comparison to the function of this gene in Drosophila. Based on the data presented, it can now be established that mirror likely has a similar selector function for posterior-wing identity in a plethora of insects.

Weaknesses:

This first version has minor terminological issues regarding the use of the terms "domains" and "compartment".

Reviewer #2 (Public Review):

Summary:

Invasive fungal infections are very difficult to treat with limited drug options. With the increasing concern of the drug resistance, developing antifungal vaccine is a high priority. In this study, authors studied the metal metabolism in Candida albicans by testing some chelators, including EDTA, to block the metal acquisition and metabolism by the fungus. Interestingly, they found EDTA treated yeast cells grew poorly in vitro and non-pathogenic in vivo in a murine model. Mice immunized by EDTA-treated Candida (CAET) were protected against challenge with wild type Candida cells. RNA-Seq analysis to survey the gene expression profile in response to EDTA treatment in vitro revealed upregulation of genes in metal homeostasis and down regulation of ribosome biogenesis. They also revealed an induction of both pro- and anti-inflammatory cytokines involved in Th1, Th2 and Th17 host immune response in response to CAET immunization. Overall, this is an interesting study with a translational potential.

Strengths:

The main strength of the report is that authors identified a potential whole cell live vaccine strain that can provide a full protection against candidiasis. Abundant data both on in vitro phenotype, gene expression profile and host immune response have been presented.

Weaknesses:

A weakness is that the immune mechanism of CAET mediated host protection remain unclear. The immune data is somewhat confusing. Authors only checked cytokines and chemokines in blood. The immune response in infected tissues and antibody response may be investigated.

Another potential concern is that using live wild type Candida cells treated with EDTA may still have chance to evolve and become infectious, considering that these treated cells still proliferate in vivo. Some of the gene regulation profiles may be transit and subjected to reverse, adding to the safety concern.

deneme

I have experienced this firsthand for many months, until my laptop got away and I failed to stay productive due to my own limiting beliefs and stupidity... Now it's hard to get back into the lifestyle of the great again.

"The worst day working on your own goals is still better than the best day working on someone else's." -- Dan Koe

100X goals force one to filter action... Impossible goals = Mental Clarity of the HIGHEST degree.

100X come from vision which in turn comes from future identity (future-self)

Gamify one's life to get progress if necessary. Integrate into systems.

Reviewer #1 (Public Review):

This study makes a substantial contribution to our understanding of the molecular evolutionary dynamics of microbial genomes by proposing a model that incorporates relatively frequent adaptive reversion mutations. In many ways, this makes sense from my own experience with evolutionary genomic data of microbes, where reversions are surprisingly familiar as evidence of the immense power of selection in large populations.

One criticism is the reliance on one major data set of B. fragilis to test fits of these models, but this is relatively minor in my opinion and can be caveated by discussion of other relevant datasets for parallel investigation.

Another point is that this problem isn't as new as the manuscript indicates, see for example https://journals.asm.org/doi/10.1128/aem.02002-20.

Nonetheless, the paper succeeds by both developing theory and offering concrete parameters to illustrate the magnitudes of the problems that distinguish competing ideas, for example, the risk of mutational load posed in the absence of frequent back mutation.

Reviewer #1 (Public Review):

(1) Napthylamine (1NA), an industrial reagent used in the manufacturing of dyes and pesticides is harmful to humans and the environment. In the current manuscript, the authors report the successful isolation of a Pseudomonas strain from a former naphthylamine manufacturing site that is capable of degrading 1NA. Using genetic and enzymatic analysis they identified the initial stages of 1NA degradation and the enzymes responsible for downstream processing of 1,2-dihydroxynapthalene and Salicylate. The authors determined the molecular structure of NpaA1, the first enzyme in the pathway responsible for glutamylation of 1NA. NpaA1 has a border substrate specificity compared to previously characterized enzymes involved in aromatic amine degradation. They carried out structural comparison of NpaA1 with glutamine synthase structures, alfa-fold models of similar enzymes and put forth hypothesis to explain the broad substrate specificity of NpaA1.

The manuscript is well written and easy to understand. The authors carried out careful genetic analysis to identify the genes/enzymes responsible for degradation of 1NA to catechol. They characterized the first enzyme in the pathway, NpaA1 which is responsible glutamylation of 1NA. and determined the molecular structure of apo-NpaA1, NpaA1 - AMPPNP complex and Npa1 - ADP - Met-Sox-P complex using X-ray crystallography.<br /> The proposed mechanism of broad substrate specificity of NpaA1, however, is based on comparison of 1NA docked NpaA1 structure with St-GS (Glutamate synthase) and Alphafold2 predicted model of AtdA1 from an aniline degrading strain of Acinetobacter sp. Lack of molecular structure or mutational studies to back the proposed mechanism makes it difficult to agree with the proposed mechanism.

I.e., 3f+1 is not suitable for open peer-to-peer systems.

Reviewer #1 (Public Review):

This study offers good evidence pointing to a genetic basis for Arabidopsis thaliana's response to elevated CO2 (eCO2) levels and its subsequent impact on the leaf ionome. The natural variation analyses in the study support the hypothesis that genetic factors, rather than local adaptation, guide the influence of eCO2 on the ionome of rosette leaves in Arabidopsis.

Comments on current version:

I appreciate the revisions and the effort the authors have made.

Most of the abstract now accurately reflects the results and methods. It would be nice to have a few more technical details in the abstract, such as:<br /> * What was the CO2 level?<br /> * Which gene was identified?

I still have a problem with this sentence:

"The elevation of atmospheric CO2 leads to a decline in plant mineral content, which might pose a significant threat to food security in the coming decades."

The authors provide a wide range of published studies that support this statement. I fully agree that this is what the literature suggests. However, I think the literature has asked the wrong question.

In general, these studies addressed the question: Given no time for adaptation, do plants grown under high CO2 have a different mineral composition? The answer is yes.

But a more important question is: Can plants and food crops adapt in time? I believe the strength of this study is that it tests this, and it suggests that the answer is yes. I also think there is a lot of unpublished results and greenhouse breeding success that supports the contention that most plants can adapt to the CO2.

"The artificial elevation of atmospheric CO2 leads to a physiological response and decline in plant mineral content, which might pose a significant threat to food security in the coming decades if plants cannot adapt."

It needs to be made clear throughout the paper when high CO2 levels lead to low mineral composition. These are all artificial manipulations without allowing the plants to adapt to the new environment.

"The elevation of atmospheric CO2 concentration leads to a decline in the mineral composition of C3 plants (Gojon et al., 2023)." - this is well supported in artificial environments.

Do wild plants have fewer minerals in their leaves today compared to plants in 1950? This would be great evidence and framing for this experiment.

Crop plants having lower nitrogen and different mineral compositions over time is substantially a product of breeders initially increasing inputs and then, over the last decade, selecting for higher input efficiency.

At the end of the introduction or the beginning of the results, please define why the CO2 level was chosen and its context as being at the high end of current predictions.

"According to the literature, this results in a 20-25% reduction in vitamin C or lycopene and requires a significantly higher nitrogen and water intake to reach expected sugar levels (Doddrell H (2023), Horticulture Research). In addition, the negative effect of elevated CO2 on tomato nutrient content seems to have significant repercussions on nutrition-health properties (Boufeldja (2023), Molecules)."

Thank you for sharing these reviews. These suggest to me that breeders favored the 80% yield bump over other traits. Either there was no breeding, or the breeding focused on other traits. It is important to mention that breeders should include mineral nutrition in their selection index while they maximize yield. Simpler breeding strategies can sometimes heavily favor one trait over others, but cattle breeders today regularly use selection indices that incorporate weights for two dozen traits.

This study provides nice evidence that an annual weed species is likely to be able to adapt easily to high eCO2. Whether perennial species will be able to adapt in time is clearly a topic that needs to be investigated.

Reviewer #1 (Public Review):

Summary:

Zai et al test if songbirds can recover the capacity to sing auditory targets without singing experience or sensory feedback. Past work showed that after the pitch of targeted song syllables are driven outside of birds' preferred target range with external reinforcement, birds revert to baseline (i.e. restore their song to their target). Here the authors tested the extent to which this restoration occurs in muted or deafened birds. If these birds can restore, this would suggest an internal model that allows for sensory-to-motor mapping. If they cannot, this would suggest that learning relies entirely on feedback dependent mechanisms, e.g. reinforcement learning (RL). The authors find that deafened birds exhibit moderate but significant restoration, consistent with the existence of a previously under-appreciated internal model in songbirds.

Strengths:

The experimental approach of studying vocal plasticity in deafened or muted birds is innovative, technically difficult and perfectly suited for the question of feedback-independent learning. The finding in Figure 4 that deafened birds exhibit subtle but significant plasticity toward restoration of their pre-deafening target is surprising and important for the songbird and vocal learning fields, in general.

In this revision, the authors suitably addressed confusion about some statistical methods related to Fig. 4, where the main finding of vocal plasticity in deafened birds was presented.

There remain minor issues in the presentation early in the results section and in Fig. 4 that should be straightforward to clarify in the revision.

Reviewer #1 (Public Review):

Summary:

This work identified new NMD inhibitors and tested them for cancer treatment, based on the hypothesis that inhibiting NMD could lead to the production of cancer neoantigens from the stabilized mutant mRNAs, thereby enhancing the immune system's ability to recognize and kill cancer cells. Key points of the study include:

• Development of an RNA-seq based method for NMD analysis using mixed isogenic cells that express WT or mutant transcripts of STAG2 and TP53 with engineered truncation mutations.

• Application of this method for a drug screen and identified several potential NMD inhibitors.

• Demonstration that one of the identified compounds, LY3023414, inhibits NMD by targeting the SMG1 protein kinase in the NMD pathway in cultured cells and mouse xenografts.

• Due to the in vivo toxicity observed for LY3023414, the authors developed 11 new SMG1 inhibitors (KVS0001-KVS0011) based on the structures of the known SMG1 inhibitor SMG1i-11 and the SMG1 protein itself.

• Among these, KVS0001 stood out for its high potency, excellent bioavailability, and low toxicity in mice. Treatment with KVS0001 caused NMD inhibition and increased presentation of neoantigens on MHC-I molecules, resulting in the clearance of cancer cells in vitro by co-cultured T cells and cancer xenografts in mice by the immune system.

These findings support the strategy of targeting the NMD pathway for cancer treatment and provide new research tools and potential lead compounds for further exploration.

Strengths:

The RNA-seq-based NMD analysis, using isogenic cell lines with specific NMD-inducing mutations, represents a novel approach for the high-throughput identification of potential NMD modulators or genetic regulators. The effectiveness of this method is exemplified by the identification of a new activity of AKT1/mTOR inhibitor LY3023414 in inhibiting NMD.

The properties of KVS0001 described in the manuscript as a novel SMG1 inhibitor suggest its potential as a lead compound for further testing the NMD-targeting strategies in cancer treatment. Additionally, this compound may serve as a useful research tool.

The results of the in vitro cell killing assay and in vivo xenograft experiments in both immuno-proficient and immune-deficient mice indicate that inhibiting NMD could be a viable therapeutic strategy for certain cancers.

Weaknesses:

The authors did not address the potential effects of NMD/SMG1 inhibitors on RNA splicing. Given that the transcripts of many RNA-binding proteins are natural targets of NMD, inhibiting NMD could significantly alter splicing patterns. This, in turn, might influence the outcomes of the RNA-seq-based method for NMD analysis and result interpretation.

While the RNA-seq-based approach offers several advantages for analyzing NMD, the effects of NMD/SMG1 inhibitors observed through this method should be confirmed using established NMD reporters. This step is crucial to rule out the possibility that mutations in STAG2 or TP53 affect NMD in cells, as well as to address potential clonal variations between different engineered cell lines.

The results from the SMG1/UPF1 knockdown and SMG1i-11 experiments presented in Figure 3 correlate with the effects seen for LY3023414, but they do not conclusively establish SMG1 as the direct target of LY3023414 in NMD inhibition. An epistatic analysis with LY3023414 and SMG1-knockdown is needed.

Reviewer #2 (Public Review):

Summary:<br /> The authors used four datasets spanning 30 countries to examine funding success and research quality score for various disciplines. They examined whether funding or research quality score were influenced by majority gender of the discipline and whether these affected men, women, or both within each discipline. They found that disciplines dominated by women have lower funding success and research quality score than disciplines dominated by men. These findings, are surprising because even the men in women-dominated fields experienced lower funding success and research quality score.

Strengths:<br /> - The authors utilized a comprehensive dataset covering 30 countries to explore the influence of the majority gender in academic disciplines on funding success and research quality scores.<br /> - Findings suggest a systemic issue where disciplines with a higher proportion of women have lower evaluations and funding success for all researchers, regardless of gender.<br /> - The manuscript is notable for its large sample size and the diverse international scope, enhancing the generalizability of the results.<br /> - The work accounts for various factors including age, number of research outputs, and bibliometric measures, strengthening the validity of the findings.<br /> - The manuscript raises important questions about unconscious bias in research evaluation and funding decisions, as evidenced by lower scores in women-dominated fields even for researchers that are men.<br /> - The study provides a nuanced view of gender bias, showing that it is not limited to individuals but extends to entire disciplines, impacting the perception and funding and quality or worth of research.<br /> - This work underscores the need to explore motivations behind gender distribution across fields, hinting at deep-rooted societal and institutional barriers.<br /> - The authors have opened a discussion on potential solutions to counter bias, like adjusting funding paylines or anonymizing applications, or other practical solutions.<br /> - While pointing out limitations such as the absence of data from major research-producing countries, the manuscript paves the way for future studies to examine whether its findings are universally applicable.

Weaknesses:<br /> - The study does not provide data on the gender of grant reviewers or stakeholders, which could be critical for understanding potential unconscious bias in funding decisions. These data are likely not available; however, this could be discussed. Are grant reviewers in fields dominated by women more likely to be women?<br /> - There could be more exploration into whether the research quality score is influenced by inherent biases towards disciplines themselves, rather than only being gender bias.<br /> - The manuscript should discuss how non-binary gender identities were addressed in the research. There is an opportunity to understand the impact on this group.<br /> - A significant limitation is absence of data from other major research-producing countries like China and the United States, raising questions about the generalizability of the findings. How comparable are the findings observed to these other countries?<br /> - The motivations and barriers that drive gender distribution in various fields could be expanded on. Are fields striving to reach gender parity through hiring or other mechanisms?<br /> - The authors could consider if the size of funding awards correlates with research scores, potentially overlooking a significant factor in the evaluation of research quality. Presumably there is less data on smaller 'pilot' funds and startup funds for disciplines where these are more common. Would funding success follow the same trend for these types of funds?<br /> - The language used in the manuscript at times may perpetuate bias, particularly when discussing "lower quality disciplines," which could influence the reader's perception of certain fields.<br /> - The manuscript does not clarify how many gender identities were represented in the datasets or how gender identity was determined, potentially conflating gender identity with biological sex.

Reviewer #1 (Public Review):

Summary:

The authors provide very compelling evidence that the lateral septum (LS) engages in theta cycle skipping.

Strengths:

The data and analysis is highly compelling regarding the existence of cycle skipping.

Comments on the revised version:

All previous recommendations were addressed in this revision.

Reviewer #1 (Public Review):

Summary:

The pituitary gonadotropins, FSH and LH, are critical regulators of reproduction. In mammals, synthesis and secretion of FSH and LH by gonadotrope cells are controlled by the hypothalamic peptide, GnRH. As FSH and LH are made in the same cells in mammals, variation in the nature of GnRH secretion is thought to contribute to the differential regulation of the two hormones. In contrast, in fish, FSH and LH are produced in distinct gonadotrope populations and may be less (or differently) dependent on GnRH than in mammals. In the present manuscript, the authors endeavored to determine whether FSH may be independently controlled by a distinct peptide, cholecystokinin (CCK), in zebrafish.

Strengths:

The authors demonstrated that the CCK receptor is enriched in FSH-producing relative to LH-producing gonadotropes, and that genetic deletion of the receptor leads to dramatic decreases in gonadotropin production and gonadal development in zebrafish. Also, using innovative in vivo and ex vivo calcium imaging approaches, they show that LH- and FSH-producing gonadotropes preferentially respond to GnRH and CCK, respectively. Exogenous CCK also preferentially stimulated FSH secretion ex vivo and in vivo.

Weaknesses:

The concept that there may be a distinct FSH-releasing hormone (FSHRH) has been debated for decades. As the authors suggest that CCK is the long-sought FSHRH (at least in fish), they must provide data that convincingly leads to such a conclusion. In my estimation, they have not yet met this burden. In particular, they show that CCK is sufficient to activate FSH-producing cells, but have not yet demonstrated its necessity. Their one attempt to do so was using fish in which they inactivated the CCK receptor using CRISPR-Cas9. While this manipulation led to a reduction in FSH, LH was affected to a similar extent. As a result, they have not shown that CCK is a selective regulator of FSH. Moreover, they do not yet demonstrate that the effects observed reflect the loss of the receptor's function in gonadotropes, as opposed to other cell types. It also is not clear whether the phenotypes of the fish reflect perturbations in pituitary development vs. a loss of CCK receptor function in the pituitary later in life. Ideally, the authors would attempt to block CCK signaling in adult fish that develop normally. For example, if CCK receptor antagonists are available, they could be used to treat fish and see whether and how this affects FSH vs. LH secretion.

In the Discussion, the authors suggest that CCK, as a satiety factor, may provide a link between metabolism and reproduction. This is an interesting idea, but it is not supported by the data presented. That is, none of the results shown link metabolic state to CCK regulation of FSH and fertility. Absent such data, the lengthy discussion of the link is speculative and not fully merited.

Also in the Discussion, the authors argue that "CCK directly controls FSH cells by innervating the pituitary gland and binding to specific receptors that are particularly abundant in FSH gonadotrophs." However, their imaging does not demonstrate innervation of FSH cells by CCK terminals (e.g., at the EM level). Moreover, they have not demonstrated the binding of CCK to these cells. Indeed, no CCK receptor protein data are shown. The calcium responses of FSH cells to exogenous CCK certainly suggest the presence of functional CCK receptors therein; but, the nature of the preparations (with all pituitary cell types present) does not demonstrate that CCK is acting directly in these cells. Indeed, the asynchrony in responses of individual FSH cells to CCK (Figure 4) suggests that not all cells may be activated in the same way. Contrast the response of LH cells to GnRH, where the onset of calcium signaling is similar across cells (Figure 3). Finally, as the authors note in the Discussion, the data presented do not enable them to conclude that the endogenous CCK regulating FSH (assuming it does) is from the brain as opposed to other sources (e.g., the gut).

Reviewer #1 (Public Review):

Summary:

In Drosophila melanogaster, ITP has functions on feeding, drinking, metabolism, excretion, and circadian rhythm. In the current study, the authors characterized and compared the expression of all three ITP isoforms (ITPa and ITPL1&2) in the CNS and peripheral tissues of Drosophila. An important finding is that they functionally characterized and identified Gyc76C as an ITPa receptor in Drosophila using both in vitro and in vivo approaches. In vitro, the authors nicely confirmed that the inhibitory function of recombinant Drosophila ITPa on MT secretion is Gyc76C-dependent (knockdown Gyc76C specifically in two types of cells abolished the anti-diuretic action of Drosophila ITPa on renal tubules). They also used a combination of multiple approaches to investigate the roles of ITPa and Gyc76C on osmotic and metabolic homeostasis modulation in vivo. They revealed that ITPa signaling to renal tubules and fat body modulates osmotic and metabolic homeostasis via Gyc76C.

Furthermore, they tried to identify the upstream and downstream of ITP neurons in the nervous system by using connectomics and single-cell transcriptomic analysis. I found this interesting manuscript to be well-written and described. The findings in this study are valuable to help understand how ITP signals work on systemic homeostasis regulation. Both anatomical and single-cell transcriptome analysis here should be useful to many in the field.

Strengths:

- The question (what receptors of ITPa in Drosophila) that this study tries to address is important. The authors ruled out the Bombyx ITPa receptor orthologs as potential candidates. They identified a novel ITP receptor by using phylogenetic, anatomical analysis, and both in vitro and in vivo approaches.

- The authors exhibited detailed anatomical data of both ITP isoforms and Gyc76C (in the main and supplementary figures), which helped audiences understand the expression of the neurons studied in the manuscript.

- They also performed connectomes and single-cell transcriptomics analysis to study the synaptic and peptidergic connectivity of ITP-expressing neurons. This provided more information for better understanding and further study on systemic homeostasis modulation.

Weaknesses:

In the discussion section, the authors raised the limitations of the current study, which I mostly agree with, such as the lack of verification of direct binding between ITPa and Gyc76C, even though they provided different data to support that ITPa-Gyc76C signaling pathway regulates systemic homeostasis in adult flies.

Reviewer #1 (Public Review):

This is an interesting, informative, and well-designed study that combines theoretical and experimental methodologies to tackle the phenomenon of higher-resolution structures/substructures in model biomolecular condensates.

The authors have adequately addressed my previous concerns.

Reviewer #1 (Public Review):

Summary:

Thakare et al propose a gravimetric method to evaluate feeding from solid food in Drosophila adults that can be used to evaluate the nutritional impact of high-fat food.

Strengths:

This method is new and fills a gap in the methods used in Drosophila research.

Weaknesses:

The data presented address a number of questions that are mainly interesting for people needing to reproduce such experiments. The work could be improved by being presented within a broader scope.

Reviewer #1 (Public Review):

Summary:

In the manuscript submission by Zhao et al. entitled, "Cardiac neurons expressing a glucagon-like receptor mediate cardiac arrhythmia induced by high-fat diet in Drosophila" the authors assert that cardiac arrhythmias in Drosophila on a high-fat diet are due in part to adipokinetic hormone (Akh) signaling activation. High-fat diet induces Akh secretion from activated endocrine neurons, which activate AkhR in posterior cardiac neurons. Silencing or deletion of Akh or AkhR blocks arrhythmia in Drosophila on a high-fat diet. Elimination of one of two AkhR-expressing cardiac neurons results in arrhythmia similar to a high-fat diet.

Strengths:

The authors propose a novel mechanism for high-fat diet-induced arrhythmia utilizing the Akh signaling pathway that signals to cardiac neurons.

Weaknesses:

Major comments:

(1) The authors state, "Arrhythmic pathology is rooted in the cardiac conduction system." This assertion is incorrect as a blanket statement on arrhythmias. There are certain arrhythmias that have been attributable to the conduction system, such as bradycardic rhythms, heart block, sinus node reentry, inappropriate sinus tachycardia, AV nodal reentrant tachycardia, bundle branch reentry, fascicular ventricular tachycardia, or idiopathic ventricular fibrillation to name a few. However the etiological mechanism of many atrial and ventricular arrhythmias, such as atrial fibrillation or substrate-based ventricular tachycardia, are not rooted in the conduction system. The introduction should be revised to reflect a clear focus on atrial fibrillation (AF). In addition, AF susceptibility is known to be modulated by autonomic tone, which is topically relevant to this manuscript.

(2) The authors state that "HFD led to increased heartbeat and an irregular rhythm." In representative examples shown, HFD resulted in pauses, slower heart rate, and increased irregularity in rhythm but not consistently increased heart rate (Figures 1B, 3A, and 4C). Based on the cited work by Ocorr et al (https://doi.org/10.1073/pnas.0609278104), Drosophila heart rate is highly variable with periods of fast and slow rates, which the authors attributed to neuronal and hormonal inputs. Ocorr et al then describe the use of "semi-intact" flies to remove autonomic input to normalize heart rate. Were semi-intact flies used? If not, how was heart rate variability controlled? And how was heart rate "increase" quantified in high-fat diet compared to normal-fat diet? Lastly, how does one measure "arrhythmia" when there is so much heart rate variability in normal intact flies?

(3) The authors state, "to test whether the HFD-induced increase in Akh in the APC affects APC neuron activity, we used CaLexA (https://doi.org/10.3109/01677063.2011.642910)." According to the reference, CaLexA is a tool to map active neurons and would not indicate, as the authors state, whether Akh affects APC neuron activity specifically. It is equally possible that APC neurons may be activated by HFD and produce more Akh. Please clarify this language.

(4) Are the AkhR+ neurons parasympathetic or sympathetic? Please provide additional experimentation that characterizes these neurons. The AkhR+ neurons appear to be anti-arrhythmic. Please expand the discussion to include a working hypothesis of the overall findings on Akh, AkhR, and AkhR+ neurons.

(5) The authors state, "Heart function is dependent on glucose as an energy source." However, the heart's main energy source is fatty acids with minimal use of glucose (doi: 10.1016/j.cbpa.2006.09.014). Glucose becomes more utilized by cardiomyocytes under heart failure conditions. Please amend/revise this statement.

Reviewer #1 (Public Review):

Summary:

The manuscript by Jang et al. describes the application of new methods to measure the localization of GTP-binding signaling proteins (G proteins) on different membrane structures in a model mammalian cell line (HEK293). G proteins mediate signaling by receptors found at the cell surface (GPCRs), with evidence from the last 15 years suggesting that GPCRs can induce G-protein mediated signaling from different membrane structures within the cell, with variation in signal localization leading to different cellular outcomes. While it has been clearly shown that different GPCRs efficiently traffic to various intracellular compartments, it is less clear whether G proteins traffic in the same manner, and whether GPCR trafficking facilitates "passenger" G protein trafficking. This question was a blind spot in the burgeoning field of GPCR localized signaling in need of careful study, and the results obtained will serve as an important guidepost for further work in this field. The extent to which G proteins localize to different membranes within the cell is the main experimental question tested in this manuscript. This question is pursued through two distinct methods, both relying on genetic modification of the G-beta subunit with a tag. In one method, G-beta is modified with a small fragment of the fluorescent protein mNG, which combines with the larger mNG fragment to form a fully functional fluorescent protein to facilitate protein trafficking by fluorescent microscopy. This approach was combined with the expression of fluorescent proteins directed to various intracellular compartments (different types of endosomes, lysosome, endoplasmic reticulum, Golgi, mitochondria) to look for colocalization of G-beta with these markers. These experiments showed compelling evidence that G-beta co-localizes with markers at the plasma membrane and the lysosome, with weak or absent co-localization for other markers. A second method for measuring localization relied on fusing G-beta with a small fragment from a miniature luciferase (HiBit) that combines with a larger luciferase fragment (LgBit) to form an active luciferase enzyme. Localization of G-beta (and luciferase signal) was measured using a method known as bystander BRET, which relies on the expression of a fluorescent protein BRET acceptor in different cellular compartments. Results using bystander BRET supported findings from fluorescence microscopy experiments. These methods for tracking G protein localization were also used to probe other questions. The activation of GPCRs from different classes had virtually no impact on the localization of G-beta, suggesting that GPCR activation does not result in the shuttling of G proteins through the endosomal pathway with activated receptors.

Strengths:

The question probed in this study is quite important and, in my opinion, understudied by the pharmacology community. The results presented here are an important call to be cognizant of the localization of GPCR coupling partners in different cellular compartments. Abundant reports of endosomal GPCR signaling need to consider how the impact of lower G protein abundance on endosomal membranes will affect the signaling responses under study.

The work presented is carefully executed, with seemingly high levels of technical rigor. These studies benefit from probing the experimental questions at hand using two different methods of measurement (fluorescent microscopy and bystander BRET). The observation that both methods arrive at the same (or a very similar) answer inspires confidence about the validity of these findings.

Weaknesses:

The rationale for fusing G-beta with either mNG2(11) or SmBit could benefit from some expansion. I understand the speculation that using the smallest tag possible may have the smallest impact on protein performance and localization, but plenty of researchers have fused proteins with whole fluorescent proteins to provide conclusions that have been confirmed by other methods. Many studies even use G proteins fused with fluorescent proteins or luciferases. Is there an important advantage to tagging G-beta with small tags? Is there evidence that G proteins with full-size protein tags behave aberrantly? If the studies presented here would not have been possible without these CRISPR-based tagging approaches, it would be helpful to provide more context to make this clearer. Perhaps one factor would be interference from newly synthesized G proteins-fluorescent protein fusions en route to the plasma membrane (in the ER and Golgi).

As noted by the authors, they do not demonstrate that the tagged G-beta is predominantly found within heterotrimeric G protein complexes. If there is substantial free G-beta, then many of the conclusions need to be reconsidered. Perhaps a comparison of immunoprecipitated tagged G beta vs immunoprecipitated supernatant, with blotting for other G protein subunits would be informative.

Additional context and questions:

(1) There exists some evidence that certain GPCRs can form enduring complexes with G-beta-gamma (Pubmed: 23297229, 27499021). That would seem to offer a mechanism that would enable receptor-mediated transport of G protein subunits. It would be helpful for the authors to place the findings of this manuscript in the context of these previous findings since they seem somewhat contradictory.

(2) There is some evidence that GaS undergoes measurable dissociation from the plasma membrane upon activation (see the mechanism of the assay in Pubmed: 35302493). It seems possible that G-alpha (and in particular GaS) might behave differently than the G-beta subunit studied here. This is not entirely clear from the discussion as it now stands.

(3) The authors say "The presence of mNG-b1 on late endosomes suggested that some G proteins may be degraded by lysosomes". The mechanism of lysosomal degradation by proteins on the outside of the lysosome is not clear. It would be helpful for the authors to clarify.

(4) Although the authors do a good job of assessing G protein dilution in endosomal membranes, it is unclear how this behavior compares to the measurement of other lipid-anchored proteins using the same approach. Is the dilution of G proteins what we would expect for any lipid-anchored protein at the inner leaflet of the plasma membrane?

Reviewer #1 (Public Review):

Summary:

In this study, the authors show that a long-non coding RNA lncDACH1 inhibits sodium currents in cardiomyocytes by binding to and altering the localization of dystrophin. The authors use a number of methodologies to demonstrate that lncDACH1 binds to dystrophin and disrupt its localization to the membrane, which in turn downregulates NaV1.5 currents. Knockdown of lncDACH1 upregulates NaV1.5 currents. Furthermore, in heart failure, lncDACH1 is shown to be upregulated which suggests that this mechanism may have pathophysiological relevance.

Strengths:

(1) This study presents a novel mechanism of Na channel regulation which may be pathophysiologically important.

(2) The experiments are comprehensive and systematically evaluate the physiological importance of lncDACH1.

Reviewer #1 (Public Review):

Wang, He et al have constructed comprehensive single nucleus atlas for the gills of the deep sea Bathymodioline mussels, which possess intracellular symbionts that provide a key source of carbon and allow them to live in these extreme environments. They provide annotations of the different cell states within the gills, shedding light on how multiple cell types cooperate to give rise to the emergent functions of the composite tissues and the gills as a whole. They pay special attention to characterizing the bacteriocyte cell populations and identifying sets of genes that may play a role in their interaction with the symbiotes.

Wang, He et al sample mussels from 3 different environments: animals from their native methane rich environment, animals transplanted to a methane-poor environment to induce starvation and animals that have been starved in the methane-poor environment and then moved back to the methane-rich environment. They demonstrated that starvation had the biggest impact on bacteriocyte transcriptomes. They hypothesize that the up-regulation of genes associated with lysosomal digestion leads to the digestion of the intracellular symbiont during starvation, while the non-starved and reacclimated groups more readily harvest the nutrients from symbiotes without destroying them. Further work exploring the differences in symbiote populations between ecological conditions will further elucidate the dynamic relationship between host and symbiote. This will help disentangle specific changes in transcriptomic state that are due to their changing interactions with the symbiotes from changes associated with other environmental factors.

This paper makes available a high quality dataset that is of interest to many disciplines of biology. The unique qualities of this non-model organism and collection of conditions sampled make it of special interest to those studying deep sea adaptation, the impact of environmental perturbation on Bathymodioline mussels populations, and intracellular symbiotes. The authors also use a diverse array of tools to explore and validate their data.

Reviewer #1 (Public Review):

Summary:

Federer et al. tested AAVs designed to target GABAergic cells and parvalbumin-expressing cells in marmoset V1. Several new results were obtained. First, AAV-h56D targeted GABAergic cells with >90% specificity, and this varied with serotype and layer. Second, AAV-PHP.eB.S5E2 targeted parvalbumin-expressing neurons with up to 98% specificity. Third, the immunohistochemical detection of GABA and PV was attenuated near viral injection sites.

Strengths:

Vormstein-Schneider et al. (2020) tested their AAV-S5E2 vector in marmosets by intravenous injection. The data presented in this manuscript are valuable in part because they show the transduction pattern produced by intraparenchymal injections, which are more conventional and efficient.

Weaknesses:

The conclusions regarding the effects of serotype are based on data from single injection tracks in a single animal. I understand that ethical and financial constraints preclude high throughput testing, but these limitations do not change what can be inferred from the measurements. The text asserts that "...serotype 9 is a better choice when high specificity and coverage across all layers are required". The data presented are consistent with this idea but do not make a strong case for it.

A related criticism extends to the analysis of injection volume on viral specificity. Some replication was performed here, but reliability across injections was not reported. My understanding is that individual ROIs were treated as independent observations. These are not biological replicates (arguably, neither are multiple injection tracks in a single animal, but they are certainly closer). Idiosyncrasies between animals or injections (e.g. if one injection happened to hit one layer more than another) could have substantial impacts on the measurements. It remains unclear which results regarding injection volume or serotype would hold up had a large number of injections been made into a large number of marmosets.

Reviewer #1 (Public Review):

Summary:

In this work, Qiu and colleagues examined the effects of preovulatory (i.e., proestrous or late follicular phase) levels of circulating estradiol on multiple calcium and potassium channel conductances in arcuate nucleus kisspeptin neurons. Although these cells are strongly linked to a role as the "GnRH pulse generator," the goal here was to examine the physiological properties of these cells in a hormonal milieu mimicking late proestrus, the time of the preovulatory GnRH-LH surge. Computational modeling is used to manipulate multiple conductances simultaneously and support a role for certain calcium channels in facilitating a switch in firing mode from tonic to bursting. CRISPR knockdown of the TRPC5 channel reduced overall excitability, but this was only examined in cells from ovariectomized mice without estradiol treatment. The patch clamp experiments are comprehensive and overall solid but a direct demonstration of the role of these conductances in being necessary for surge generation (or at least having a direct physiological consequence on surge properties) is lacking, substantially reducing the impact of the findings.

Strengths:

(1) Examination of multiple types of calcium and potassium currents, both through electrophysiology and molecular biology.

(2) Focus on arcuate kisspeptin neurons during the surge is relatively conceptually novel as the anteroventral periventricular nucleus (AVPV) kisspeptin neurons have received much more attention as the "surge generator" population.

(3) The modeling studies allow for direct examination of manipulation of single and multiple conductances, whereas the electrophysiology studies necessarily require examination of each current in isolation. The construction of an arcuate kisspeptin neuron model promises to be of value to the reproductive neuroendocrinology field.

Weaknesses:

(1) The novelty of some of the experiments needs to be clarified. This reviewer's understanding is that prior experiments largely used a different OVX+E2 treatment paradigm mimicking periods of low estradiol levels, whereas the present work used a "high E2" treatment model. However, Figures 10C and D are repeated from a previous publication by the same group, according to the figure legend. Findings from "high" vs. "low" E2 treatment regimens should be labeled and clearly separated in the text. It would also help to have direct comparisons between results from low E2 and high E2 treatment conditions.

(2) In multiple places, links are made between the changes in conductances and the transition from peptidergic to glutamatergic neurotransmission. However, this relationship is never directly assessed. The data that come closest are the qPCR results showing reduced Tac2 and increased Vglut2 mRNA, but in the figure legend, it appears that these results are from a prior publication using a different E2 treatment regimen.

(3) Similarly, no recordings of arcuate-AVPV glutamatergic transmission are made so the statements that Kiss1ARH neurons facilitate the GnRH surge via this connection are still only conjecture and not supported by the present experiments.

(4) Figure 1 is not described in the Results section, and is only tenuously connected to the statement in the introduction in which it is cited. The relevance of panels C and D is not clear. In this regard, much is made of the burst firing pattern that arises after E2 treatment in the model, but this burst firing pattern is not demonstrated directly in the slice electrophysiology examples.

(5) In Figure 3, it would be preferable to see the raw values for R1 and R2 in each cell, to confirm that all cells were starting from a similar baseline. In addition, it is unclear why the data for TTA-P2 is not shown, or how many cells were recorded to provide this finding.

(6) In Figure 5, panel C lists 11 cells in the E2 condition but panel E lists data from 37 cells. The reason for this discrepancy is not clear.

(7) In all histogram figures, it would be preferable to have the data for individual cells superimposed on the mean and SEM.

(8) The CRISPR experiments were only performed in OVX mice, substantially limiting interpretation with respect to potential roles for TRPC5 in shaping arcuate kisspeptin neuron function during the preovulatory surge.

(9) Furthermore, there are no demonstrations that the CRISPR manipulations impair or alter the LH surge.

(10) The time of day of slice preparation and recording needs to be specified in the Methods.

Reviewer #1 (Public Review):

The manuscript by Zhao et al describes the identification of RAPSYN, a NEDD8 E3 ligase previously studied for its role in acetylcholine receptor clustering and neuromuscular junction formation, as a factor promoting the stabilisation of the BCR-ABL oncogene in Chronic Myeloid Leukemia (CML) cells. The authors have identified that NEDDylation of BCR-ABL by RAPSYN antagonises its poly-ubiquitin and subsequent proteasome-based degradation. Knocking down RAPSYN with shRNA led to increased poly-ubiquitination and faster turnover of BCR-ABL. Furthermore, they describe that SRC-dependent phosphorylation of RAPSYN facilitates its NEDD8-ligase activity.

The authors' findings are primarily rooted in a series of well-conducted in vitro experiments using two CML cell lines, K562 and MEG-01. They have performed some further validations using primary CML samples, which have strengthened their claims.

The author's initial discoveries have come from interrogating a number of publicly available gene expression datasets, both microarray-based and RNA-seq, which revealed that RAPSYN is increased at the protein level but that RNA levels are not different between healthy and CML samples. This is a very interesting observation which warrants further future investigation.

The conclusions of this revised manuscript are broadly supported by the data and the analyses. It also describes novel findings that can spur future studies, both into the basic cellular biology of CML as well as into potential new therapeutic strategies.

Comments on revised version:

I thank the authors for addressing my concerns in the initial review. The revised manuscript with additional data is much stronger.

Reviewer #1 (Public Review):

In this study, the researchers aimed to investigate the cellular landscape and cell-cell interactions in cavernous tissues under diabetic conditions, specifically focusing on erectile dysfunction (ED). They employed single-cell RNA sequencing to analyze gene expression patterns in various cell types within the cavernous tissues of diabetic individuals. The researchers identified decreased expression of genes associated with collagen or extracellular matrix organization and angiogenesis in several cell types, including fibroblasts, chondrocytes, myofibroblasts, valve-related lymphatic endothelial cells, and pericytes. They also discovered a newly identified marker, LBH, that distinguishes pericytes from smooth muscle cells in mouse and human cavernous tissues. Furthermore, the study revealed that pericytes play a role in angiogenesis, adhesion, and migration by communicating with other cell types within the corpus cavernosum. However, these interactions were found to be significantly reduced under diabetic conditions. The study also investigated the role of LBH and its interactions with other proteins (CRYAB and VIM) in maintaining pericyte function and highlighted their potential involvement in regulating neurovascular regeneration. Overall, the manuscript is well-written and the study provides novel insights into the pathogenesis of ED in patients with diabetes and identifies potential therapeutic targets for further investigation.

Comments on revised version:

All my concerns have been properly addressed.

Reviewer #1 (Public Review):

Summary:

This paper by Watanabe et al described an expression system that can express the paired heavy and light chains of IgG antibodies from single cell B cells. In addition, they used FACS sorting for specific antigens to screen/select the specific populations for more targeted cloning of mAb genes. By staining with multiple antigens, they were able to zoom in to cross-reactive antibodies.

Strengths:

A highly efficient process that combines selection/screening with dua expression of both antibody chains. It is particularly suitable for the isolation of cross-reactive antibodies against conserved epitopes of different antigens, such as surface proteins of related viruses.

Weaknesses:

(1) The overall writing is very difficult to follow and the authors need to work on significant re-writing.

(2) The paper in its current form really lacks detail and it is NOT possible for readers to repeat or follow their methods. For example: a) It is not clear whether the authors checked the serum to see if the mice were producing antibodies before they sacrificed them to harvest spleen/blood i.e. using ELISA? b) How long after administration of the second dose were the mice sacrificed? c) What cell types are taken for single B cell sorting? Splenocytes or PBMC? These are just some of the questions which need to be addressed.

(3) According to the authors, 77 clones were sorted from the PR8+ and H2+ double positive quadrant. It is surprising that after transfection and re-analysing of bulk antibody presenting EXPI cells on FACS, only 13 clones (or 8 clones? - unclear) seemed to be truly cross-reactive. If that is the case, the approach is not as efficient as the authors claimed.

Reviewer #1 (Public Review):

This is a very nice study of Belidae weevils using anchored phylogenomics that presents a new backbone for the family and explores, despite a limited taxon sampling, several evolutionary aspects of the group. The phylogeny is useful to understand the relationships between major lineages in this group and preliminary estimation of ancestral traits reveals interesting patterns linked to host-plant diet and geographic range evolution. I find that the methodology is appropriate, and all analytical steps are well presented. The paper is well-written and presents interesting aspects of Belidae systematics and evolution. The major weakness of the study is the very limited taxon sampling which has deep implications for the discussion of ancestral estimations.

Reviewer #1 (Public Review):

Summary:

Páramo et al. used 3D geometric morphometric analyses of the articulated femur, tibia, and fibula of 17 macronarian taxa (known to preserve these three skeletal elements) to investigate morphological changes that occurred in the hind limb through the evolutionary history of this sauropod clade. A principal components analysis was completed to understand the distribution of the morphological variation. A supertree was constructed to place evolutionary trends in morphological variation into phylogenetic context, and hind limb centroid size was used to investigate potential relationships between skeletal anatomy and gigantism. The majority of the results did not yield statistically significant differences, but they did identify interesting shape-change trends, especially within subclades of Titanosauria. Many previous studies have attempted to elucidate a link between wide-gauge posture and gigantism, which in this study Páramo et al. investigate among several titanosaurian subclades. They propose that morphologies associated with wide-gauge posture arose in parallel with increasing body size among basal members of Macronaria and that this connection became less significant once wide-gauge posture was acquired within Titanosauria. The authors also suggest that other biomechanical factors influenced the independent evolution of subclades within Titanosauria and that these influences resulted in instances of convergent evolution. Therefore, they infer that, overall, wide-gauge posture was not significantly correlated with gigantism, though some morphological aspects of hind limb skeletal anatomy appear to have been associated with gigantism. Their work also supports previous findings of a decrease in body size within Titanosauriformes (which they found to be not significant with shape variables but significant with Pagel's lambda). Collectively, their results support and build on previous work to elucidate more specifics on the evolution of this enigmatic clade. Further study will show if their hypotheses stand or if the inclusion of additional specimens and taxa yields alternative results.

Strengths:

Páramo et al. were diligent in their efforts to digitize and prepare specimens for this study while also minimizing user bias. Their previous work provided a strong platform for this study, specifically for their robust methodology. Between their supplemental files (which include details about specimen digitization and preparation) and the main body of the manuscript, the authors fully provide their results in detailed tables and figures. Their conclusions on evolutionary trends within Titanosauria are reasonably well supported (see weaknesses below) and they provide important details that enhance our understanding of the evolution of this clade and complement previous findings. Their discussion of links between morphology and various biomechanical adaptations is important, and future studies can use these results to investigate such biomechanical adaptations. The trends they identify within the subclades of Titanosauria are very interesting and highlight the diversity of this clade. It is possible that additional investigations of the evolution of these subclades could unite findings in sauropod myology and biomechanics, each of which has been suggested to vary among titanosaurian taxa without a clear phylogenetic or evolutionary distribution. The authors suggest that certain common morphologies arose via convergent evolution among titanosaurian subclades, such as members of Colossosauria exhibiting morphologies more similar to basal titanosaurians than derived saltasaurines. While this conclusion about convergent evolution is not well explained, only future testing will determine if this hypothesis remains supported. Additionally, the authors discuss the influence of uncertainty on the phylogenetic position of some taxa, and this reminds readers to view their findings as tentative trends that may be illuminated through further quantitative analyses. If one accepts the use of hind limb centroid size as a reliable approximation of body size (see concerns in Weaknesses below) then their data also support a hypothesis of decreasing body size through titanosaurian evolution (with PC 2 further differentiating small titanosaurian taxa from one another), providing an opportunity for future analyses to further investigate these interesting trends.

Weaknesses:

Several sentences throughout the manuscript could benefit from citations. For example, the discussion of using hind limb centroid size as a proxy for body mass has no citations attributed. This should be cited or described as a new method for estimating body mass with data from extant taxa presented in support of this relationship. This particular instance is a very important point to include supporting documentation because the authors' conclusions about evolutionary trends in body size are predicated on this relationship.

An additional area of concern is the lack of any discussion of taphonomic deformation in Section 3.3 Caveats of This Study, the results, or the methods. The authors provide a long and detailed discussion of taphonomic loss and how this study does a good job of addressing it; however, taphonomic deformation to specimens and its potential effects on the ensuing results were not addressed at all. Hedrick and Dodson (2013) highlight that, with fossils, a PCA typically includes the effects of taphonomic deformation in addition to differences in morphology, which results in morphometric graphs representing taphomorphospaces. For example, in this study, the extreme negative positioning of Dreadnoughtus on PC 2 (which the authors highlight as "remarkable") is almost certainly the result of taphonomic deformation to the distal end of the holotype femur, as noted by Ullmann and Lacovara (2016).

The authors investigated 17 taxa and divided them into 9 clades, with only Titanosauria and Lithostrotia including more than two taxa (and four clades are only represented by one taxon). While some of these clades represent the average of multiple individuals, the small number of plotted taxa can only weakly support trends within Titanosauria. If similar general trends could be found when the taxa are parsed into fewer, more inclusive clades, it would support and strengthen their claims. Of course, the authors can only study what is preserved in the fossil record, and titanosaurian remains are often highly fragmentary; these deficiencies should therefore not be held against the authors. They clearly put effort and thought into their choices of taxa to include in this study, but there are limitations arising from this low sample size that inherently limit the confidence that can be placed on their conclusions, and this caveat should be more clearly discussed. Specifically, the authors note that their dataset contains many lithostrotians, but they do not discuss unevenness in body size sampling. As neither their size-category boundaries nor the taxa which fall into each of them are clearly stated, the reader must parse the discussion to glean which taxa are in each size category. It should be noted that the authors include both Jainosaurus and Dreadnoughtus as 'large' taxa even though the latter is estimated to have been roughly five times the body mass of the former, making Dreadnoughtus the only taxon included in this extreme size category. The effects that this may have on body size trends are not discussed. Additionally, few taxa between the body masses of Jainosaurus and Dreadnoughtus have been included even though the hind limbs of several such macronarians have been digitized in prior studies (such as Diamantinasaurus and Giraffititan; Klinkhamer et al. 2018). Also, several members of Colossosauria are more similar in general body size to Dreadnoughtus than Jainosaurus, but unfortunately, they do not preserve a known femur, tibia, and fibula, so the authors could not include them in this study. Exclusion of these taxa may bias inferences about body size evolution, and this is a sampling caveat that could have been discussed more clearly. Future studies including these and other taxa will be important for further evaluating the hypotheses about macronarian evolution advanced by Páramo et al. in this study.

Reviewer #1 (Public Review):

Summary:

In the paper entitled "PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis", the authors sought to determine the role of HSCB, a known regulator of Iron sulfur cluster transfer, in the generation of erythrocytes and megakaryocytes. They utilized a human primary cell model of hematopoietic differentiation to identify a novel mechanism whereby HSCB is necessary for activation of erythroid and megakaryocytic gene expression through regulation of the nuclear localization of FOG-1, a essential transcription co-regulator of the GATA transcription factors. Their work establishes this novel regulatory axis as a mechanism by which cytokine signaling through EPO-R and MPL drives the lineage-specification of hematopoietic progenitors to erythrocytes and megakaryocytes, respectively.

Impact:

The major impact of this work is in a greater understanding of how cytokine signaling through EPO/TPO function to promote lineage specification of hematopoietic stem/progenitor cells. While the major kinase cascades downstream of the EPO/TPO receptors have been elucidated, how those cascades effect gene expression to promote a specific differentiation program is poorly understood. For this work, we now understand that nuclear localization of FOG is a critical regulatory node by which EPO/TPO signaling is required to launch FOG-dependent gene expression. However, these cytokine receptors have many overlapping and redundant targets, so it still remains to be elucidated how signaling through the different receptors promotes divergent gene expression programs. Perhaps similar regulatory mechanisms exist for other lineage-specifying transcription factors.

Strengths:

The authors use two different cellular models of erythroid differentiation (K562 and human primary CD34+ cells) to elucidate the multi-factorial mechanism controlling FOG-1 nuclear localization. The studies are well-controlled and rigorously establish their mechanism through complementary approaches. The differentiation effects are established through cell surface marker expression, protein expression, and gene expression analyses. Novel protein interactions discovered by proteomics analyses were validated through bi-directional co-IP experiments in multiple experimental systems. Protein cellular localization findings are supported by both immunofluorescence and cell fractionation immunoblot analyses. The robustness of their experimental findings gives great confidence for the likelihood that the methods and findings can be reproduced in future work based on their conclusions.

Weaknesses:

The one unexplained step in this intricately described mechanism is how HSCB functions to promote TACC3 degradation. It appears that the proteasome is involved since MG-132 reverses the effect of HSCB deficiency, but no other details are provided. Does HSCB target TACC3 for ubiquitination somehow? Future studies will be required to understand this portion of the mechanism.

One weakness of the study design is that no in vivo experiments are conducted. The authors comment that the HSCB mouse phenotype is too dramatic to permit studies of erythropoiesis in vivo; however, a conditional approach could have been pursued.<br /> It should also be noted that a previous study had already shown that TACC3 regulates the nuclear localization of FOG-1, so this portion of the mechanism is not entirely novel. However, the role of HSCB and the proteasomal degradation of TACC3 is entirely novel to my knowledge.

Reviewer #1 (Public Review):

Summary:

This is an interesting study that performs scRNA-Seq on infected and uninfected wounds. The authors sought to understand how infection with E. faecalis influences the transcriptional profile of healing wounds. The analysis demonstrated that there is a unique transcriptional profile in infected wounds with specific changes in macrophages, keratinocytes, and fibroblasts. They also speculated on potential crosstalk between macrophages and neutrophils and macrophages and endothelial cells using NicheNet analysis and CellChat. Overall the data suggest that infection causes keratinocytes to not fully transition which may impede their function in wound healing and that the infection greatly influenced the transcriptional profile of macrophages and how they interact with other cells.

Strengths:

It is a useful dataset to help to understand the impact of wound infection on transcription of specific cell types. The analysis is very thorough in terms of transcriptional analysis and uses a variety of techniques and metrics.

Weaknesses:

Some drawbacks of the study are the following. First the fact that it only has two mice per group, and only looks at one time point after wounding decreases the impact of the study. Wound healing is a dynamic and variable process so understanding the full course of the wound healing response would be very important to understand the impact of infection on the healing wound. The analysis has been bolstered by applying a cross-entropy test on the integrated dataset and to ensure robustness of the datasets (Fig S1F). Including unwounded skin in the scRNA-Seq would also lend a lot more significance to this study. However, this was technically challenging due to constraints with the number of immune cells in unwounded skin as described in the limitations section. Another drawback of the study is that mouse punch biopsies are very different than human wounds as they heal primarily by contraction instead of re-epithelialization like human wounds. The authors mitigated this somewhat be extracting the incisional parts of the wound. So while the conclusions are generally supported the scope of the work is somewhat limited.

Reviewer #1 (Public Review):

The current manuscript revisits previous reports in the literature. The human Pannexin 1 channel is regulated by phosphorylation at two residues by Src kinase. From this series of experiments, the authors conclude that PANX-1 is not phosphorylated at these residues.

The biggest strength of the manuscript is the comprehensiveness of the approach. The authors recapitulate prior experiments in the literature and also add a series of new, orthogonal experiments that all examine the claim of PANX-1 phosphorylation. The breadth of the reported experiments extends over multiple cell lines and protein constructs, in vitro purified proteins, mass spec, different phosphorylation detection reagents and antibodies, and functional electrophysiology assays that show that the addition of Src does not impact gating. The combined weight of all these data strongly suggests that the field should re-examine the claim that PANX-1 is regulated by phosphorylation at Y199 and Y309.

Another strength is that the authors go beyond simply showing that the antibodies do not recognize phosphorylated PANX-1. They also provide potential mechanisms for how the antibodies may be misleading. Both antibodies recognize phosphorylated Src-1. In the case of anti-PANX1-pY308, the authors provide solid mutagenesis evidence that the antibody also weakly recognizes a non-phosphorylated epitope of PANX1 in the same region as the tyrosine. This helps make a convincing case.

Such experiments, while not glamorous, have great practical importance for developing an accurate understanding of how Pannexin channels are regulated.

Reviewer #2 (Public Review):

In this revised manuscript Aguillon and collaborators convincingly demonstrating that CLK is required for free-running behavioral rhythms under constant conditions in the Cnidarian Nematostella. The results also convincingly show that CLK impacts rhythmic gene expression in this organism. This original work thus demonstrates that CLK was recruited very early during animal evolution in the circadian clock mechanism to optimize behavior and gene expression with the time-of-day.

Reviewer #1 (Public Review):

Summary:

The authors analyzed the bacterial colonization of human sperm using 16S rRNA profiling. Patterns of microbiota colonization were subsequently correlated with clinical data, such as spermiogram analysis, the presence of reactive oxygen species (ROS), and DNA fragmentation. The authors identified three main clusters dominated by Streptococcus, Prevotella, and Lactobacillus & Gardnerella, respectively, which aligns with previous observations. Specific associations were observed for certain bacterial genera, such as Flavobacterium and semen quality. Overall, it is a well-conducted study that further supports the importance of the seminal microbiota.

Strengths:

- The authors performed the analysis on 223 samples, which is the largest dataset in semen microbiota analysis so far.<br /> - Inclusion of negative controls to control contaminations.<br /> - Inclusion of a positive control group consisting of men with proven fertility.

Weaknesses:<br /> - The manuscript needs comprehensive proofreading for language and formatting. In many instances, spaces are missing or not required.<br /> - Could the authors explore correlation network analyses to get additional insights into the structure of different clusters?<br /> - The GitHub link is not correct.<br /> - It is not possible to access the dataset on ENA.<br /> - Add the graphs obtained with decontam analysis as a supplementary figure.<br /> - There is nothing about the RPL group in the results section, while the authors discuss this issue in the introduction. What about the controls with proven fertility?<br /> - While correctly stated in the title, the term microbiota should be used throughout the manuscript instead of "microbiome"

Reviewer #1 (Public Review):

Summary:

This manuscript set out to identify selective inhibitors of the pyridoxal phosphatase (PDXP). Previous studies had demonstrated improvements in cognition upon removal of PDXP, and here the authors reveal that this correlates with an increase in pyridoxal phosphate (PLP; PDXP substrate and an active coenzyme form of vitamin B6) with age. Since several pathologies are associated with decreased vitamin B6, the authors propose that PDXP is an attractive therapeutic target in the prevention/treatment of cognitive decline. Following high throughput and secondary small molecule screens, they identify two selective inhibitors. They follow up on 7, 8 dihydroxyflavone (DHF). Following structure-activity relationship and selectivity studies, the authors then solve a co-crystal structure of 7,8 DHF bound to the active site of PDXP, supporting a competitive mode of PDXP inhibition. Finally, they find that treating hippocampal neurons with 7,8 DHF increases PLP levels in a WT but not PDXP KO context. The authors note that 7,8 DHF has been used in numerous rodent neuropathology models to improve outcomes. 7, 8 DHF activity was previously attributed to activation of the receptor tyrosine kinase TrkB, although this appears to be controversial. The present study raises the possibility that it instead/also acts through modulation of PLP levels via PDXP, and is an important area for future work.

Strengths:

The strengths of the work are in the comprehensive, thorough, and unbiased nature of the analyses revealing the potential for therapeutic intervention in a number of pathologies.

Weaknesses:

Potential weaknesses include the poor solubility of 7,8 DHF that might limit its bioavailability given its relatively low potency (IC50= 0.8 uM), which was not improved by SAR. The solubility issues of 7,8 DHF have been discussed at length in the authors' response to Reviewer #3. In particular, the solubility of 7,8 DHF has been found to be variable due to the concentration and buffer conditions. The 7,8 DHF compound has an extended residence time and the co-crystal structure could aid the design of more potent molecules and would be of interest to those in the pharmaceutical industry. The images related to crystal structure have been improved with additional structural analysis of PDXP in a complex of 7,8-DHF (see revised Figure 3).

Reviewer #1 (Public Review):

Summary:

Bendzunas, Byrne et al. explore two highly topical areas of protein kinase regulation in this manuscript. Firstly, the idea that Cys modification could regulate kinase activity. The senior authors have published some standout papers exploring this idea of late, and the current work adds to the picture of how active site Cys might have been favoured in evolution to serve critical regulatory functions. Second, BRSK1/2 are understudied kinases listed as part of the "dark kinome" so any knowledge of their underlying regulation is of critical importance to advancing the field.

Strengths:

In this study, the author pinpoints highly-conserved, but BRSK-specific, Cys residues as key players in kinase regulation. There is a delicate balance between equating what happens in vitro with recombinant proteins relative to what the functional consequence of Cys mutation might be in cells or organisms, but the authors are very clear with the caveats relating to these connections in their descriptions and discussion. Accordingly, by extension, they present a very sound biochemical case for how Cys modification might influence kinase activity in cellular environs.

Comments on revised version:

The authors have satisfactorily addressed my concerns.

Reviewer #2 (Public Review):

Summary:

The authors try to establish that there is an Abeta-dependent loss of nuclear pores early in Alzheimer's disease. To do so the authors compared different NUP proteins and assessed their function by analyzing nuclear leakage and resistance to induction of nuclear damage and the associated necroptosis. The authors use a mouse knockin for hAPP with familial Alzheimer's mutations to model amyloidosis related to Alzheimer's disease. Treatment with an inhibitor of beta-amyloid production partially rescued the loss of nuclear pore proteins in young KI neurons, implicating beta-amyloid in Nuclear Pore dysfunction, a mechanism already described in other neurodegenerative diseases but not in Alzheimer's disease.

Comments on revised version:

Upon careful review, some of the critical concerns raised have yet to be fully addressed (the authors did not adequately address the two points of my public review or 5 of my 7 recommendation points), particularly regarding the effects of maturation stage or age. This has negatively impacted my initial enthusiasm for the paper, as the current approach does not fully capture the role of nuclear pore dysfunction in Alzheimer's disease, which is intimately dependent on aging. Here are specific recommendations for further revision:

(1) The manuscript would benefit from a clearer acknowledgement of the limitations concerning the effects of maturation or age. I recommend removing mentions of the effect of time, for example:

(i) Line 1 "4: "By using brain tissues and primary neurons cultured from App KI and wildtype (WT) mice, we observed a loss of NPCs in neuronal nuclei over time. "

(ii) Line 20 "13: "Similarly, in neuron cocultures, there was an 20 increase in intracellular Aβ levels over WT neurons that parallels the reduction of NUPs as neurons 21 mature from DIV "-28. "

(2) The subheading in the Discussion section, "Age-dependent decline in nuclear function during normal aging and in AD," could be more accurately retitled "Nuclear function decline" in AD" to avoid suggesting age dependence without the requisite data.

(3) Because primary neurons differentiate, mature, and age with time in culture, they are required to control for the developmental stage of your cultures. Please include the control data that would support cultures maturation stage, such as staining for axodendritic markers (e.g., MAP2), glial cell distribution (e.g., GFAP), and the balance of excitatory vs. inhibitory neuronal subpopulations (e.g., Gad65). This data is crucial for substantiating the culture conditions and the resulting interpretations.

Reviewer #1 (Public Review):

The study by Longhurst et al. investigates the mechanisms of chemoresistance and chemosensitivity towards three compounds that inhibit cell cycle progression: camptothecin, colchicine, and palbociclib. Genome-wide genetic screens were conducted using the HAP1 Cas9 cell line, revealing compound-specific and shared pathways of resistance and sensitivity. The researchers then focused on novel mechanisms that confer resistance to palbociclib, identifying PRC2.1. Genetic and pharmacological disruption of PRC2.1 function, but not related PRC2.2, leads to resistance to palbociclib. The researchers then show that disruption of PRC2.1 function (for example, by MTF2 deletion), results in locus-specific changes in H3K27 methylation and increases in D-type cyclin expression. It is suggested that increased expression of D-type cyclins results in palbociclib resistance.

Strengths:

The results of this study are interesting and contribute insights into the molecular mechanisms of CDK4/6 inhibitors. Importantly, while CDK4/6 inhibitors are effective in the clinic, tumour recurrence is very high due to acquired resistance.

Weaknesses:

A key resistance mechanism is Rb loss, so it is important to understand if resistance conferred by PRC2.1 loss is mediated by Rb, and whether restoration of PRC2.1 function in Rb-deplete cells results in renewed palbociclib sensitivity. It is also important to understand the clinical implications of the results presented. The inclusion of these data would significantly improve the paper. However, besides some presentation issues and typos as described below, it is my opinion that the results are robust and of broad interest.

Major questions:

(1) Is the resistance to CDK4/6 inhibition conferred by mutation of MTF2 mediated by Rb?

(2) Are mutations in PRC2.1 found in genetic analyses of tumour samples in patients with acquired resistance?

Reviewer #1 (Public Review):

Summary:

In this manuscript, Tung and colleagues identify Calreticulin as a repressor of ATF6 signaling using a CRISPR screen and characterize the functional interaction between ATF6 and CALR.

Strengths:

The manuscript is well written and interesting with an innovative experimental design that provides some new mechanistic insight into ATF6 regulation as well as crosstalk with the IRE1 pathway. The methods used were fit for purpose and reasonable conclusions were drawn from the data presented. Findings are novel and bring together glycoprotein quality control and activation of one sensor of the UPR. This is a novel perspective on how the integration of ER homeostasis signals could be sensed in the ER.

Weaknesses:

Several points remain to be documented to support the authors' model.

Reviewer #1 (Public Review):

Summary:

The manuscript "Engineering of PAClight1P78A: A High-Performance Class-B1 GPCR-Based Sensor for PACAP1-38" by Cola et al. presents the development of a novel genetically encoded sensor, PAClight1P78A, based on the human PAC1 receptor. The authors provide a thorough in vitro and in vivo characterization of this sensor, demonstrating its potential utility across various applications in life sciences, including drug development and basic research.

The diverse methods to validate PAClight1P78A demonstrate a comprehensive approach to sensor engineering by combining biochemical characterization with in vivo studies in rodent brains and zebrafish. This establishes the sensor's biophysical properties (e.g., sensitivity, specificity, kinetics, and spectral properties) and demonstrates its functionality in physiologically relevant settings. Importantly, the inclusion of control sensors and the testing of potential intracellular downstream effects such as G-protein activation underscore a careful consideration of specificity and biological impact.

Strengths:

The fundamental development of PAClight1P78A addresses a significant gap in sensors for Class-B1 GPCRs. The iterative design process -starting from PAClight0.1 to the final PAClight1P78A variant - demonstrates compelling optimization. The innovative engineering results in a sensor with a high apparent dynamic range and excellent ligand selectivity, representing a significant advancement in the field. The rigorous in vitro characterization, including dynamic range, ligand specificity, and activation kinetics, provides a critical understanding of the sensor's utility. Including in vivo experiments in mice and zebrafish larvae demonstrates the sensor's applicability in complex biological systems.

Weaknesses:

The manuscript shows that the sensor fundamentally works in vivo, albeit in a limited capacity. The titration curves show sensitivity in the nmol range at which endogenous detection might be possible. However, perhaps the sensor is not sensitive enough or there are not any known robust paradigms for PACAP release. A more detailed discussion of the sensors's limitations, particularly regarding in vivo applications and the potential for detecting endogenous PACAP release, would be helpful.

There are several experiments with an n=1 and other low single-digit numbers. I assume that refers to biological replicates such as mice or culture wells, but it is not well defined. n=1 in experimental contexts, particularly in Figure 1, raises significant concerns about the exact dynamic range of the sensor, data reproducibility, and the robustness of conclusions drawn from these experiments. Also, ROI for cell cultures, like in Figure 1, is not well defined. The methods mentioned ROIs were manually selected, which appears very selective, and the values in Figure 1c become unnecessarily questionable. The lack of definition for "ROI" is confusing. Do ROIs refer to cells, specific locations on the cell membrane, or groups of cells? It would be best if the authors could use unbiased methods for image analysis that include the majority of responsive areas or an explanation of why certain ROIs are included or excluded.

Reviewer #1 (Public Review):

Summary:

In this study, James Lee, Lu Bai, and colleagues use a multifaceted approach to investigate the relationship between transcription factor condensate formation, transcription, and 3D gene clustering of the MET regulon in the model organism S. cerevisiae. This study represents a second clear example of inducible transcriptional condensates in budding yeast, as most evidence for transcriptional condensates arises from studies of mammalian systems. In addition, this study links the genomic location of transcriptional condensates to the potency of transcription of a reporter gene regulated by the master transcription factor contained in the condensate. The strength of evidence supporting these two conclusions is strong. Less strong is evidence supporting the claim that Met4-containing condensates mediate the clustering of genes in the MET regulon.

Strengths:

The manuscript is for the most part clearly written, with the overriding model and specific hypothesis being tested clearly explained. Figure legends are particularly well written. An additional strength of the manuscript is that most of the main conclusions are supported by the data. This includes the propensity of Met4 and Met32 to form puncta-like structures under inducing conditions, formation of Met32-containing LLPS-like droplets in vitro (within which Met4 can colocalize), colocalization of Met4-GFP with Met4-target genes under inducing conditions, enhanced transcription of a Met3pr-GFP reporter when targeted within 1.5 - 5 kb of select Met4 target genes, and most impressively, evidence that several MET genes appear to reposition under transcriptionally inducing conditions. The latter is based on a recently reported novel in vivo methylation assay, MTAC, developed by the Bai lab.

Weaknesses:

My principal concern is that the authors fail to show convincing evidence for a key conclusion, highlighted in the title, that nuclear condensates per se drive MET gene clustering. Figure 4E demonstrates that Met4 molecules, not condensates per se, are necessary for fostering distant cis and trans interactions between MET6 and three other Met4 targets under -met inducing conditions. In addition, the paper would be strengthened by discussing a recent study conducted in yeast that comes to many of the same conclusions reported here, including the role of inducible TF condensates in driving 3D genome reorganization (Chowdhary et al, Mol. Cell 2022).

Other concerns:

(1) A central premise of the study is that the inducible formation of condensates underpins the induction of MET gene transcription and MET gene clustering. Yet, Figure 1 suggests (and the authors acknowledge) that puncta-like Met4-containing structures pre-exist in the nuclei of non-induced cells. Thus, the transcription and gene reorganization observed is due to a relatively modest increase in condensate-like structures. Are we dealing with two different types of Met4 condensates? (For example, different combinations of Met4 with its partners; Mediator- or Pol II-lacking vs. Mediator- or Pol II-containing; etc.?) At the very least, a comment to this effect is necessary.

(2) Using an in vitro assay, the authors demonstrate that Met4 colocalizes with Met32 LLPS droplets (Figure 2F). Is the same true in vivo - that is, is Met32 required for Met4 condensation? This could be readily tested using auxin-induced degradation of Met32. Along similar lines, the claim that Met32 is required for MET gene clustering (line 250) requires auxin-induced degradation of this protein.

(3) The authors use a single time point during -met induction (2 h) to evaluate TF clustering, transcription (mRNA abundance), and 3D restructuring. It would be informative to perform a kinetic analysis since such an analysis could reveal whether TF clustering precedes transcriptional induction or MET gene repositioning. Do the latter two phenomena occur concurrently or does one precede the other?

(4) Based on the MTAC assay, MET13 does not appear to engage in trans interactions with other Met4 targets, whereas MET6 does (Figures 4C and 4E). Does this difference stem from the greater occupancy of Met4 at MET6 vs. MET13, greater association of another Met co-factor with the chromatin of MET6 vs. MET13, or something else?

Reviewer #1 (Public Review):

There is an undisputable need for better in vitro models recapitulating steatotic liver diseases. This article is from a group of well-known stem cell experts that use human induced pluripotent stem cells (hiPSCs) to build a multicellular steatosis model in vitro. While the model is strong for testing hepatocytes responses, it falls short on translational aspects as well as on non-parenchymal liver cells.

(1) The authors should use the new nomenclature for the disease, MASLD / MASH, as proposed by the scientific societies (Rinella ME, et al. J Hepatol. 2023; 79(6):1542-1556. PMID: 37364790).

(2) There has been a similar approach by the Takebe group (Ouchi R, et al., Cell Metab. 2019; 30(2):374-384, PMID: 31155493). What is different in this model?

(3) The work is very technical and does neither provide any new mechanistic insights nor does it test any new interventions. I do see the clear technical advance in the long-term culture. However, I do not see that this system would allow modelling true "chronic" changes in MASLD, e.g. steatohepatitis and/or fibrosis.

(4) While I am very convinced about the validity of the "hepatocyte" component in this system, the NPC compartment is insufficient. The 3D model does certainly not contain Kupffer cells (which have very distinct characteristics from "M0" macrophages) and does not contain true HSCs (LX-2 is a very insufficient model). Also, the model lacks flow conditions, which does not allow to factor in pathogenic signals from the circulation / portal vein (e.g. gut-liver axis). This will only allow very limited insights into the crosstalk between hepatocytes and NPCs.

(5) The translational value of this model remains unclear to me. The scRNA-seq data should be meticulously compared to sc/snRNA-seq data from human MASLD livers at different stages to understand, what this system is able to model (maybe very early stages of steatosis?).

(6) The study lacks a "use case" to study interventions, e.g. testing resmetirom or any other of the new MASLD drugs in this system.

Reviewer #1 (Public Review):

Summary:

The evolution of non-shivering thermogenesis is of fundamental importance to understand. Here, in small mammals the contractile apparatus of the muscle are shown to increase energy expenditure upon a drop in ambient temperature. Additionally, in the state of torpor, small hibernators did not show an increase in energy expenditure under the same challenge.

Strengths:

The authors have conducted a very well-planned study that has sampled the muscle of large and small hibernators from two continents. Multiple approaches were then used to identify the state of the contractile apparatus, and its energy expenditure under torpor or otherwise.

Weaknesses:

There was only one site of biopsy from the animals used (leg). As the authors state, it would be interesting to know if non-shivering thermogenesis is something that is regionally different in the animal, given the core body and distal limbs have different temperatures.

Reviewer #1 (Public Review):

The Calcium Homeostasis Modulators (CALHM) are a family of large pore channels, of which the physiological role of CALHM1 and 3 is well understood, in particular their key role in taste sensation via the release of the neurotransmitter ATP. The activation mechanism of CALHM1 involves membrane depolarization and a decrease in extracellular Ca concentration, allowing the passage of large cellular metabolites. However, the activation mechanism and physiological roles of other family members are much less well understood. Many structures of homomeric CALHM proteins have been determined, revealing distinct oligomeric assemblies despite a common transmembrane domain topology. CALHM1 and 3 have been shown functionally to form heteromeric assemblies with properties distinct from those of homomeric CALHM1. However, the structural basis of heteromeric CALHM1 and 3 remains unexplored.

In this paper, Drozdzyk et al. present an important study on the structures of heteromeric channels composed of CALHM2 and CALHM4, extending the structural understanding of the CALHM family beyond homomeric channels. The study relies primarily on cryo-EM. Despite the inherent challenges of structural determination due to the similar structural features of CALHM2 and CALHM4, the authors innovatively use synthetic nanobodies to distinguish between the subunits. Their results show a broad distribution of different heteromeric assemblies, with CALHM4 conformation similar to its homomeric form and CALHM2 conformation influenced by its proximity to CALHM4, and provide detailed insights into the interaction between CALHM2 and CALHM4.

The manuscript is well-structured and presents clear results that support the conclusions drawn. The discovery of heteromeric CALHM channels, although currently limited to an overexpressed system, represents a significant advance in the field of large-pore channels and will certainly encourage further investigation into the physiological relevance and roles of heteromeric CALHM channels.

Comments on the revised version:

I appreciate the authors' efforts to try the alternative data processing strategy. Congratulations to the authors for this interesting and important work!

Reviewer #1 (Public Review):

As outlined in my previous public review, Yeo et al. revised the current neuronal intoxication model, common to all serotypes of botulinum neurotoxins. Using a combination of genetic and imaging approaches, they demonstrate that upon internalization, BoNT/A-containing endosomes undergo retro-axonally trafficking to the neuronal soma. Within the soma, this particular serotype then traffics to the endoplasmic reticulum (ER) via the Golgi apparatus. At the ER, the SEC61 translocon complex facilitates the translocation of BoNT/A's metalloprotease domain (light chain, LC) from the ER lumen into the cytosol, where the thioredoxin reductase/thioredoxin system and HSP complexes release and refold the catalytic LC. Subsequently, the LC diffuses and cleaves SNAP25 first in the soma before reaching neurites and synapses.

Although I still acknowledge the well-executed and thoroughly analyzed genome-wide RNAi screen, I must once again highlight significant pitfalls and weaknesses in the paper due to the lack of essential controls and validations. Consequently, I suggest readers to approach the authors' findings with caution, as they may be limited to the combination of one specific cellular model and genetic engineering tools. During the revision process, authors declined to conduct additional experiments that could have strengthened their main conclusions. These include, but are not limited to:

(1) Investigating weather in the newly generated cell line Red-SNAPR, the GFP fragment produced upon toxin cleavage degrades more rapidly in the soma compared to axon terminals, possibly due to differences in proteasome activity in these two compartments.

(2) Validating toxin cleavage activity in the soma before reaching synapses by conducting an additional and more physiological approach, a time course experiment using native BoNT/A and staining BoNT/A-cleaved SNAP25 with specific antibodies.

(3) Assessing whether the addition of mNG1-11 to the LC affects the translocation process itself and quantifying the mean fluorescence intensity (MFI) per cell, taking into consideration the amount of HA-tagged Cyt-mG1-10, which appears predominantly expressed in the cytosol and less detected in neurites. This raises the question of potential bias toward the cell soma in this assay.

(4) Validating major hits (e.g., VPS34 and Sec61) by performing WB or IF analysis to test the cleavage of endogenous SNAP25.

Additionally, during the revision process, the authors raised concerns about the level of scrutiny applied by this reviewer, particularly in comparison to the seminal study of Lilia K. Koriazova & Mauricio Montal published in Nature Structural Biology (PMID: 12459720). In this 2003 paper, Montal's lab pioneered the use of single-channel recordings and substrate proteolysis analysis to reconstitute the translocation of BoNT/A light chain protease across an artificial lipid bilayer via the channel formed by its heavy chain. The authors highlighted that, when converting the experimental conditions from the aforementioned paper into molarity, it appears that the cis compartment was loaded with 10−8 M BoNT/A, and the reported translocated protease activity (measured by substrate cleavage) is equivalent to 10−17 M. This implies that only about 1 LC molecule in 100 million has crossed the membrane. The calculation performed by authors is indeed accurate. However, readers should be informed about another piece of information present in the same paper that might help them to clarify this important point. Koriazova & Montal, by discussing this experiment, have pointed out that this value (10−17 M) corresponds to ≈3600 LC molecules, a number closed to the maximum number of channels that can be formed under the used experimental conditions. Indeed, from the same paper, quotation: 'This number is in close agreement with the maximum number of channels inserted in the bilayer under the assay condition, ≈2000 (Fig. 3a), as estimated from macroscopic membrane conductance ∼1 × 105 pS and γ = 50 pS measured in 0.1 M KCl'. Another aspect that Yeo et al. forgot to mention in their rebuttal letter is that the system used by Koriazova & Montal lacks any chaperones in the trans compartment. Nowadays, we know that upon translocation, the refolding of the L chain is aided by Hsp90 (Azarnia Tehran et al., Cellular microbiology, 2017). Keeping this in mind, is not unrealistic to hypothesize that the number of LC molecules calculated more than 22 years ago by Koriazova & Montal (in an indirect way by checking SNAP25 cleavage using an ELISA-based assay) might be an underestimation. Indeed, the addition of Hsp90 in their system might aid in the refolding of LC molecules that, even if they have successfully be translocated, might not cleave the substrate due to their unfolded state.

As active scientist, I understand the challenges of peer review and publication, which can often be slow and frustrating involving seemingly endless rounds of review. Therefore, I am in favor of the new eLife publishing model. Indeed, this paper has already been published as Reviewed Preprints and will soon be declared as the final Version of Record, accompanied by this public review. Having said that, I hope that the readers of this journal and future scientists will prove me wrong. I hope they will engage with this paper, providing comments, validations (which are currently missing), and citations as frequently as they did for the seminal works of Koriazova & Montal.

Reviewer #1 (Public Review):

The paper combines experiments on freely gliding cyanobacteria, buckling experiments using two-dimensional V shaped corners, and micropipette force measurements with theoretical models to study gliding forces in these organisms. The aim is to quantify these forces and use the results to perhaps discriminate between competing mechanisms by which these cells move. A large data set of possible collision events are analyzed, bucking events evaluated, and critical buckling lengths estimated. A line elasticity model is used to analyze the onset of buckling and estimate the effective (viscous type) friction/drag that controls the dynamics of the rotation that ensues post-buckling. This value of the friction/drag is compared to a second estimate obtained by consideration of the active forces and speeds in freely gliding filaments. The authors find that these two independent estimates of friction/drag correlate with each other and are comparable in magnitude. The experiments are conducted carefully, the device fabrication is novel, the data set is interesting, and the analysis is solid. The authors conclude that the experiments are consistent with the propulsion being generated by adhesion forces rather than slime extrusion. While consistent with the data, this conclusion is inferred.

Summary:

The paper addresses important questions on the mechanisms driving the gliding motility of filamentous cyanobacteria. The authors aim to understand these by estimating the elastic properties of the filaments, and by comparing the resistance to gliding under a) freely gliding conditions, and b) in post-buckled rotational states. Experiments are used to estimate the propulsion force density on freely gliding filaments (assuming over damped conditions). Experiments are combined with a theoretical model based on Euler beam theory to extract friction (viscous) coefficients for filaments that buckle and begin to rotate about the pinned end. The main results are estimates for the bending stiffness of the bacteria, the propulsive tangential force density, the buckling threshold in terms of the length, and estimates of the resistive friction (viscous drag) providing the dissipation in the system and balancing the active force. It is found that experiments on the two bacterial species yield nearly identical value of 𝑓 (albeit with rather large variations). The authors conclude that the experiments are consistent with the propulsion being generated by adhesion forces rather than slime extrusion.

Strengths of the paper:

The strengths of the paper lie in the novel experimental setup and measurements that allow for the estimation of the propulsive force density, critical buckling length, and effective viscous drag forces for movement of the filament along its contour - the axial (parallel) drag coefficient, and the normal (perpendicular) drag coefficient (I assume this is the case, since the post-buckling analysis assumes the bent filament rotates at a constant frequency). These direct measurements are important for serious analysis and discrimination between motility mechanisms.

Weaknesses:

There are aspects of the analysis and discussion that may be improved. I suggest that the authors take the following comments into consideration while revising their manuscript.

The conclusion that adhesion via focal adhesions is the cause for propulsion rather than slime protrusion, is consistent with the experimental results that the frictional drag correlates with propulsion force. At the same time, it is hard to rule out other factors that may result in this (friction) viscous drag - (active) force relationship while still being consistent with slime production. More detailed analysis aiming to discriminate between adhesion vs slime protrusion may be outside the scope of the study, but the authors may still want to elaborate on their inference. It would help if there was a detailed discussion on the differences in terms of the active force term for the focal adhesion-based motility vs the slime motility.

Can the authors comment on possible mechanisms (perhaps from the literature) that indicate how isotropic friction may be generated in settings where focal adhesions drive motility. A key aspect here would probably be estimating the extent of this adhesion patch and comparing it to a characteristic contact area. Can lubrication theory be used to estimate characteristic areas of contact (knowing the radius of the filament, and assuming a height above substrate)? If the focal adhesions typically cover areas smaller than this lubrication area, it may suggest the possibility that bacteria essentially present a flat surface insofar as adhesion is concerned, leading to transversely isotropic response in terms of the drag. Of course, we will still require the effective propulsive force to act along the tangent.

I am not sure why the authors mention that the power of the gliding apparatus is not rate limiting. The only way to verify this would be to put these in highly viscous fluids where the drag of the external fluid comes into the picture as well (if focal adhesions are on the substrate facing side, and the upper side is subject to ambient fluid drag). Also, the friction referred to here has the form of a viscous drag (no memory effect, and thus not viscoelastic or gel-like), and it is not clear if forces generated by adhesion involve other forms of drag such as chemical friction via temporary bonds forming and breaking. In quasi-static settings and under certain conditions such as separation of chemical and elastic time scales, bond friction may yield overall force proportional to local sliding velocities.

For readers from a non-fluids background, some additional discussion of the drag forces, and the forms of friction would help. For a freely gliding filament if 𝑓 is the force density (per unit length), then steady gliding with a viscous frictional drag would suggest (as mentioned in the paper) 𝑓 ∼ 𝑣! 𝐿 𝜂∥. The critical buckling length is then dependent on 𝑓 and on 𝐵 the bending modulus. Here the effective drag is defined per length. I can see from this that if the active force is fixed, and the viscous component resulting from the frictional mechanism is fixed, the critical buckling length will not depend on the velocity (unless I am missing something in their argument), since the velocity is not a primitive variable, and is itself an emergent quantity.

Reviewer #1 (Public Review):

Summary:

Tsai and Seymen et al. investigate associations between RTE expression and methylation and age and inflammation, using multiple public datasets. The concept of the study is in principle interesting, as a systematic analysis of RTE expression during human aging is lacking. Unfortunately, the reliance on expression microarray data, used to perform the core analysis of the paper places much of the study on shaky ground. The findings of the study would not be sufficiently supported until the authors validate them with more suitable methods.

Strengths:

This is a very important biological problem.

Weaknesses:

RNA microarray probes are obviously biased to genes, and thus quantifying transposon analysis based on them seems dubious. Based on how arrays are designed there should at least be partial (perhaps outdated evidence) that the probe sites overlap a protein-coding or non-coding RNA. The authors state they only used intergenic probes, but based on supplementary files, almost half of RTE probes are not intergenic but intronic (n=106 out of 264). This is further complicated by the fact that not all this small subset of probes is available in all analyzed datasets. For example, 232 probes were used for the MESA dataset but only 80 for the GTP dataset. Thus, RTE expression is quantified with a set of probes which is extremely likely to be highly affected by non-RTE transcripts and that is also different across the studied datasets. Differences in the subsets of probes could very well explain the large differences between datasets in multiple of the analyses performed by the authors, such as in Figure 2a, or 3a. It is nonetheless possible that the quantification of RTE expression performed by the authors is truly interpretable as RTE expression, but this must be validated with more data from RNA-seq. Above all, microarray data should not be the main type of data used in the type of analysis performed by the authors.

Reviewer #2 (Public Review):

Congenital cystic airway abnormalities (CPAM) are a common poorly understood disorder in airway lung development that can be fatal if not effectively treated at birth. This study by Luo and colleagues provides compelling new evidence that bone morphogenetic protein signaling in distal mesenchymal cells is required for normal mouse lung development. Genetic loss of BMP receptor in mice and in fetal mesenchymal cells causes type 2 or alveolar-like CPAM pathology. Furthermore, this is associated with changes in expression of Sox2-Sox9 suggesting defects in the proximal to distal cellularity of the lung. Interestingly, cysts are formed even when SMAD1 and 5, two major downstream effects of BMP signaling are deleted suggesting a role for non-canonical BMP signalling. Furthermore, they were independent of ablating BMP signaling in non-vascular mesenchymal cells. The findings are compelling and provide strong evidence that cystic lung development is caused by loss of non-canonical BMP signaling in mesenchymal cells. The main weakness of the paper is that it does not identify the downstream non-canonical effector of mesenchymal BMP signaling. The authors provide a plausible suggestion that it may be p38 MAPK that deserves further investigation. Despite this minor weakness, the overall findings are novel and considered important because they provide a foundation for new studies, including experiments that may produce drugs designed to prevent or treat newborn infants with CPAM.

Reviewer #1 (Public Review):

Summary:

This manuscript explores the impact of serotonin on olfactory coding in the antennal lobe of locusts and odor-evoked behavior. The authors use serotonin injections paired with an odor-evoked palp-opening response assay and bath application of serotonin with intracellular recordings of odor-evoked responses from projection neurons (PNs).

Strengths:

The authors make several interesting observations, including that serotonin enhances behavioral responses to appetitive odors in starved and fed animals, induces spontaneous bursting in PNs, directly impacts PN excitability, and uniformly enhances PN responses to odors.

Weakness:

The one remaining issue to be resolved is the theoretical discrepancy between the physiology and the behavior. The authors provide a computational model that could explain this discrepancy and provide the caveat that while the physiological data was collected from the antennal lobe, but there could be other olfactory processing stages involved. Indeed other processing stages could be the sites for the computational functions proposed by the model. There is an additional caveat which is that the physiological data were collected 5-10 minutes after serotonin application whereas the behavioral data were collected 3 hours after serotonin application. It is difficult to link physiological processes induced 5 minutes into serotonin application to behavioral consequences 3 hours subsequent to serotonin application. The discrepancy between physiology and behavior could easily reflect the timing of action of serotonin (i.e. differences between immediate and longer-term impact).

Overall, the study demonstrates the impact of serotonin on odor-evoked responses of PNs and odor guided behavior in locust. Serotonin appears to have non-linear effects including changing the firing patterns of PNs from monotonic to bursting and altering behavioral responses in an odor-specific manner, rather than uniformly across all stimuli presented.

Reviewer #1 (Public Review):

The manuscript introduces a bioinformatic pipeline designed to enhance the structure prediction of pyoverdines, revealing an extensive and previously overlooked diversity in siderophores and receptors. Utilizing a combination of feature sequence and phylogenetic approaches, the method aims to address the challenging task of predicting structures based on dispersed gene clusters, particularly relevant for pyoverdines.

Predicting structures based on gene clusters is still challenging, especially pyoverdines as the gene clusters are often spread to different locations in the genome. An improved method would indeed be highly useful, and the diversity of pyoverdine gene clusters and receptors identified is impressive.

However, so far the method basically aligns the structural genes and domains involved in pyoverdine biosynthesis and then predicts A domain specificity to predict the encoded compounds. Both methods are not particularly new as they are included in other tools such as PRISM (10.1093/nar/gkx320 ) or Sandpuma (https://doi.org/10.1093/bioinformatics/btx400) among others. The study claims superiority in A domain prediction compared to existing tools, yet the support is currently limited, relying on a comparison solely with AntiSMASH. A more extensive and systematic comparison with other tools is needed.

Additionally, in contradiction to the authors' claims, the method's applicability seems constrained to well-known and widely distributed gene clusters. The absence of predictions for new amino acids raises concerns about its generalizability to NRPS beyond the studied cases.

The manuscript lacks clarity on how the alignment of structural genes operates when dealing with multiple NRPS gene clusters on different genome contigs. How would the alignment of each BGC work?

Another critical concern is that a main challenge in NRPS structure prediction is not the backbone prediction but rather the prediction of tailoring reactions, which is not addressed in the manuscript at all, and this limitation extensively restricts the applicability of the method.

The manuscript presents a potentially highly useful bioinformatic pipeline for pyoverdine structure prediction, showcasing a commendable exploration of siderophore diversity. However, some of the claims made remain unsubstantiated. Overall, while the study holds promise, further validation and refinement are required to fulfill its potential impact on the field of bioinformatic structure prediction.

Reviewer #1 (Public Review):

The authors provided a detailed analysis of the real-time structural changes in actin filaments resulting from cofilin binding, using High-Speed Atomic Force Microscopy (HSAFM). The cofilin family controls the lifespan of actin filaments in the cells by severing the filament and promoting depolymerization. Understanding the effects of cofilin on actin filament structure is critical. It is widely acknowledged that cofilin binding significantly shortens the pitch of the actin helix. The authors previously reported (1) that this shortening extends to the unbound region of the actin filament on the pointed end side of the cluster. In this study, the authors presented substantially improved AFM images and provide detailed accounts of the dynamics observed. It was found that a minimal cofilin-binding cluster, consisting of 2-4 molecules, could induce changes in the helical parameters over one or more actin crossover repeats. Adjacent to the cofilin-binding clusters, the actin crossovers were observed to shortened within seconds, and this shortening was limited to one side of the cluster. Additionally, the phosphate binding to the actin filament was observed to stabilize the helical twist, suggesting a mechanism in which cofilin preferentially binds to ADP-bound actin filaments. These findings significantly advance our understanding of actin filament dynamics which is essential for a wide of cellular processes.<br /> However, I propose that the sections about MAD and certain parts of the discussions need substantial revisions.

MAD analysis<br /> The authors have presented findings that the mean axial distance (MAD) within actin filaments exhibits a significant dependency on the helical twist, a conclusion not previously derived despite extensive analyses through electron microscopy (EM) and molecular dynamics (MD) simulations. Notably, the MAD values span from 4.5 nm (8.5 pairs per half helical pitch, HHP) to 6.5 nm (4.5 pairs/HHP) as depicted in Figure 3C. The inner domain (ID) of actin remains very similar across C, G, and F forms(2, 3), maintaining similar ID-ID interactions in both cofilactin and bare actin filaments, keeping the identical axial distance between subunits in the both states. This suggests that the ID is unlikely to undergo significant structural changes, even with fluctuations in the filament's twist, keeping the ID-ID interactions and the axial distances. The broad range of MAD values reported poses a challenge for explanation. A careful reassessment of the MAD analysis is recommended to ensure accuracy.<br /> In determining axial distances, the authors extracted measurements from filament line profiles. It is advised to account for potential anomalies such as missing peaks or pseudo peaks, which could arise from noise interference. An example includes the observation of three peaks in HHP6 of Figure Supplement 5C, corresponding to 4.5 pairs. Peak intervals measured from the graph were 5, 11.8, 8.7, and 5.7 nm. The second region (11.8 nm) appears excessively long. If one peak is hidden in the second region, the MAD becomes 5.5 nm.

Compiling histograms of axial distances (ADs) rather than focusing solely on MAD may provide deeper insights. If the AD is too long or too short, the authors should suspect the presence of missing peaks or pseudo-peaks due to noise. If 4.4 or 5.5 pairs/HHP regions tend to contain missing peaks and 7.5-8.5 pairs/HHP regions tend to contain pseudo peaks, this may explain the MAD dependency on the helical twist.

Additionally, Figure 3E indicates a first decay constant of 0.14 seconds, substantially shorter than the frame rate (0.5 sec/frame). This suggests significant variations in line profiles between frames, attributable either to overly rapid dynamics or a low signal-to-noise ratio. Implementing running frame averages (of 2-3 frames) is recommended to distinguish between these scenarios. If the dynamics are indeed fast, the averaged frame's line profile may degrade, complicating peak identification. Conversely, if poor signal-to-noise ratio is the cause, averaging frames could facilitate peak detection. In the latter case, the authors can find the optimal number of frame averages and obtain better line profiles with fewer missing and pseudo-peaks.

Discussions<br /> The authors suggest a strong link between the C-form of actin and the formation of a short pitch helix. However, Oda et al. (3) have demonstrated that the C-form is highly unstable in the absence of cofilin binding, casting doubt on the possibility of the C-form propagating without cofilin binding. Moreover, in one strand of the cofilactin, interactions between actin subunits are limited to those between the inner domains (ID-ID interactions), which are quite similar to the interactions observed in bare actin filaments. This similarity implies that ID-ID interactions alone are insufficient to determine the helical parameters, suggesting that the presence of cofilin is essential for the formation of the short pitch helix in the cofilactin filament. Thus, crossover repeats are not necessarily shortened even if the actin form is C-form.

Narita (4) proposes that the facilitation of cofilin binding may occur through a shortening in the helix pitch, independent of a change to the C-form of actin. Furthermore, the dissociation of the D-loop from an adjacent actin subunit leads directly to the transition of actin to the G-form, which is considered the most stable configuration for the actin molecule (3).

The mechanism by which the shortened pitch propagates remains a critical and unresolved issue. It appears that this propagation is not a result of the C-form's propagation but likely involves an unidentified mechanism. Identifying and understanding this mechanism represents an essential direction for future research.

(1) K. X. Ngo et al., a, Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy. eLife 4, (2015).<br /> (2) K. Tanaka et al., Structural basis for cofilin binding and actin filament disassembly. Nature communications 9, 1860 (2018).<br /> (3) T. Oda et al., Structural Polymorphism of Actin. Journal of molecular biology 431, 3217-3228 (2019).<br /> (4) A. Narita, ADF/cofilin regulation from a structural viewpoint. Journal of muscle research and cell motility 41, 141-151 (2020).

Reviewer #1 (Public Review):

Summary:

The study provides valuable insights into the role of PfMORC in Plasmodium's epigenetic regulation, backed by a comprehensive methodological approach. The overarching goal was to understand the role of PfMORC in epigenetic regulation during asexual blood stage development, particularly its interactions with ApiAP2 TFs and its potential involvement in the regulation of genes vital for Plasmodium virulence. To achieve this, they conducted various analyses. These include a proteomic analysis to identify nuclear proteins interacting with PfMORC, a study to determine the genome-wide localization of PfMORC at multiple developmental stages, and a transcriptomic analysis in PfMORCHA-glmS knockdown parasites. Taken together, this study suggests that PfMORC is involved in chromatin assemblies that contribute to the epigenetic modulation of transcription during the asexual blood stage development.

Strengths:

The study employed a multi-faceted approach, combining proteomic, genomic, and transcriptomic analyses, providing a holistic view of PfMORC's role. The proteomic analysis successfully identified several nuclear proteins that may interact with PfMORC. The genome-wide localization offered valuable insights into PfMORC's function, especially its predominant recruitment to subtelomeric regions. The results align with previous findings on PfMORC's interaction with ApiAP2 TFs. Notably, the authors meticulously contextualized their findings with prior research adding credibility to their work.

Weaknesses:

While the study identifies potential interacting partners and loci of binding, direct functional outcomes of these interactions remain an inference. The use of the glmS ribozyme system to achieve a 50% reduction in PfMORC transcript levels makes it difficult to understand the role of PfMORC solely in terms of chromatin architecture without considering its impact on gene expression. Although assessing the overall impact of acute MORC depletion was beyond the scope of the study, it would have been informative.

Reviewer #1 (Public Review):

Summary

The authors use an elegant but somewhat artificial heterodimerisation approach to activate the isolated cytoplasmic domains of different receptor kinases (RKs) including the receptor kinase BRI1 and EFR. The developmental RK BRI1 is known to be activated by the co-receptor BAK1. Active BRI1 is then able to phosphorylate downstream substrates. The immune receptor EFR is also an active protein kinase also activated by the co-receptor BAK1. EFR however appears to have little or no kinase activity but seems to use an allosteric mechanism to in turn enable BAK1 to phosphorylate the substrate kinase BIK1. EFR tyrosine phosphorylation by BAK1 appears to trigger a conformational change in EFR, activating the receptor. Likewise, kinase activating mutations can cause similar conformational transitions in EFR and also in BAK1 in vitro and in planta.

Strengths:

I particularly liked The HDX experiments coupled with mutational analysis (Fig. 2) and the design and testing of the kinase activating mutations (Fig. 3), as they provide novel mechanistic insights into the activation mechanisms of EFR and of BAK1. These findings are nicely extended by the large-scale identification of EFR-related RKs from different species with potentially similar activation mechanisms (Fig. 5).

Weaknesses:

In my opinion, there are currently two major issues with the present manuscript. (1) The authors have previously reported that the EFR kinase activity is dispensible for immune signaling (https://pubmed.ncbi.nlm.nih.gov/34531323/) but the wild-type EFR receptor still leads to a much better phosphorylation of the BIK1 substrate when compared to the kinase inactive D849N mutant protein (Fig. 1). (2) How the active-like conformation of EFR is in turn activating BAK1 is poorly characterized, but appears to be the main step in the activation of the receptor complex. Extending the HDX analyses to resting and Rap-activated receptor complexes could be a first step to address this question, but these HDX studies were not carried out due to technical limitations.

Overall this is an interesting study that aims to advance our understanding of the activation mechanisms of different plant receptor kinases with important functions in plant immunity.

Reviewer #1 (Public Review):

(a) Summary: The present study addresses how the local abundance of metabolites impacts the biology of the tumor microenvironment. The authors enroll patients harboring kidney tumors and use freshly resected tumor material for metabolic studies. Specifically, the authors separate the adjacent normal kidney tissue from the tumor material and then harvest the interstitial fluid from the normal kidney (KIF) or the tumor (TIF) for quantitative metabolomics. The plasma samples from the patient are used for comparison. Additionally, the authors also compare metabolite levels in the plasma of patients with kidney versus lung cancer (or healthy donors) to address how specific tumor types might contribute to circulating levels of metabolites. Altogether, the authors find that the metabolite levels in the KIF and TIF, although vastly different than plasma, are largely overlapping. These findings indicate that tissue of origin appears to have a stronger role in determining the local metabolic environment of tumors than the genetics or biochemistry of the tumor itself.

(b) Strengths: The biggest strength of the current study is the use of human patient-derived samples. The cohort size (~50 patients) is relatively large, which adds to the rigor of the work. The work also relies on a small pool of metabolites that can be quantitatively measured using methods developed by the authors. Focusing on a smaller metabolic pool also likely increases the signal-to-noise ratio and enables the more rigorous determination of any underlying differences. The manuscript is well-written and highlights both the significance of the findings and also acknowledges many of the caveats. The recognition of the metabolic contributions of surrounding normal tissue as the primary driver of local nutrient abundance is a novel finding in the work, which can be leveraged in future studies.

(c) Weaknesses: The work has certain caveats, some of which have been already recognized by the authors. These include the use of steady-state metabolites and the possibility of cross-contamination of some TIF into the adjacent KIF. This study is also unable to distinguish the mechanisms driving the metabolic changes in KIF/TIF relative to circulating levels in plasma.

The relative similarity of KIF and TIF is quite surprising. However, this interpretation is presently based on sampling of only ~100 polar metabolites and ~200 lipid molecules. It is, perhaps, possible that future technological developments that enable more comprehensive quantitative metabolic profiling might distinguish between KIF and TIF composition.

In vitro tissue culture is recognized to suffer from 'non-physiological' nutrient dependencies, which are impacted by the composition of culture media. Thus, in vivo studies remain our current gold-standard in mechanistic studies of tumor metabolism. It is presently unclear whether the findings of this work will be recapitulated in any of the kidney cancer in vivo models and thus be functionally testable.

The authors have acknowledged these caveats and where possible provided textual clarifications and updated figures in their revised manuscript. Future work will be required to model these changes in animal models.

Reviewer #1 (Public Review):

The study is designed to assess the role of Syngap1 in regulating the physiology of the MGE-derived PV+ and SST+ interneurons. Syngap1 is associated with some mental health disorders, and PV+ and SST+ cells are the focus of many previous and likely future reports from studies of interneuron biology, highlighting the translational and basic neuroscience relevance of the authors' work.

Strengths of the study are using well-established electrophysiology methods and the highly controlled conditions of ex vivo brain slice experiments combined with a novel intersectional mouse line, to assess the role of Syngap1 in regulating PV+ and SST+ cell properties. The findings revealed that in the mature auditory cortex, Syngap1 haploinsufficiency decreases both the intrinsic excitability and the excitatory synaptic drive onto PV+ neurons from Layer 4. In contrast, SST+ interneurons were mostly unaffected by Syngap1 haploinsufficiency. Pharmacologically manipulating the activity of voltage-gated potassium channels of the Kv1 family suggested that these channels contributed to the decreased PV+ neuron excitability by Syngap insufficiency. These results therefore suggest that normal Syngap1 expression levels are necessary to produce normal PV+ cell intrinsic properties and excitatory synaptic drive, albeit, perhaps surprisingly, inhibitory synaptic transmission was not affected by Syngap1 haploinsufficiency.

Since the electrophysiology experiments were performed in the adult auditory cortex, while Syngap1 expression was potentially affected since embryonic stages in the MGE, future studies should address two important points that were not tackled in the present study. First, what is the developmental time window in which Syngap1 insufficiency disrupted PV+ neuron properties? Albeit the embryonic Syngap1 deletion most likely affected PV+ neuron maturation, the properties of Syngap-insufficient PV+ neurons do not resemble those of immature PV+ neurons. Second, whereas the observation that Syngap1 haploinsufficiency affected PV+ neurons in auditory cortex layer 4 suggests auditory processing alterations, MGE-derived PV+ neurons populate every cortical area. Therefore, without information on whether Syngap1 expression levels are cortical area-specific, the data in this study would predict that by regulating PV+ neuron electrophysiology, Syngap1 normally controls circuit function in a wide range of cortical areas, and therefore a range of sensory, motor and cognitive functions. These are relatively minor weaknesses regarding interpretation of the data in the present study that the authors could discuss.

Reviewer #1 (Public Review):

In this manuscript, the authors described a computational method catELMo for embedding TCR CDR3 sequences into numeric vectors using a deep-learning-based approach, ELMo. The authors applied catELMo to two applications: supervised TCR-epitope binding affinity prediction and unsupervised epitope-specific TCR clustering. In both applications, the authors showed that catELMo generated significantly better binding prediction and clustering performance than other established TCR embedding methods.

The authors have addressed all of my concerns except for one as following:

(5) GIANA's result is like

– ## TIME:2020-12-14 14:45:14|cmd: GIANA4.py|COVID_test/rawData/hc10s10.txt|IsometricDistance_Thr=7.0|thr_v=3.7|thr_s=3.3|exact=True|Vgene=True|ST=3

– ## Column Info: CDR3 aa sequence, cluster id, other information in the input file<br /> CAISDGTAASSTDTQYF 1 TRBV10-3*01 6.00384245917387e-05 0.930103216755186 COVID19:BS-EQ-0002-T1-replacement_TCRB.tsv<br /> CAISDGTAASSTDTQYF 1 TRBV10-3*01 4.34559031223066e-05 0.918135389545364 COVID19:BS-EQ-0002-T2-replacement_TCRB.tsv<br /> CANATLLQVLSTDTQYF 2 TRBV21-1*01 3.00192122958694e-05 0.878695260046097 COVID19:BS-EQ-0002-T1-replacement_TCRB.tsv<br /> CANATLLQVLSTDTQYF 2 TRBV21-1*01 1.44853010407689e-05 0.768125375525736 COVID19:BS-EQ-0002-T2-replacement_TCRB.ts<br /> ...

as in its example file at: https://raw.githubusercontent.com/s175573/GIANA/master/data/hc10s10--RotationEncodingBL62.txt

The results directly give the clustering results in the second column, and there is no direct distance metric for hierarchical clustering. Therefore, it is still not clear how the authors conducted the hierarchical clustering on GIANA's results. Did the hierarchical clustering apply to each of the original clusters on the CDR3 distances within the same original cluster?

Reviewer #1 (Public Review):

To understand the spinal locomotor circuits, we need to reveal how various types of spinal interneurons work in the circuits. So far, the general roles of the cardinal groups of spinal interneurons (dI6, V0, V1, V2a, V2b, and V3) involved in locomotion have been roughly established but not fully understood. Each group is believed to contain some clades with more detailed functional differences. However, each character and function of these clades has not yet been elucidated.

In this study, Worthy et al. investigated clades of V1 neurons that are one of the main groups of inhibitory neurons in the spinal cord. Previous reports proposed four clades (Renshaw cells, FoxP2, sp8, and pou6f2) in V1 neurons defined by the expression of transcription factors. For V1 neurons in each of the four clades, the authors investigated the birth time and showed the postnatal location in the spinal cord according to the birth time. They found FoxP2-V1 located near LMC motor neurons that project to the limb. Using genetically labeled Foxp2-V1 mice, they showed that most of the synapses of V1 neurons on the cell bodies of motor neurons were from Foxp2-V1 and Renshaw cells. Furthermore, a higher proportion of Foxp2-V1 synapses is observed on LMC motor neurons than on axial motor neurons. They proposed that Foxp2-V1, which represents 60% of V1, can be further classified according to the expression of transcription factors Otp and Foxp4.

These results will be helpful for future analyses of the development and function of V1 neurons. In particular, the discovery of strong synaptic connections between Foxp2-V1 and LMC motor neurons will be beneficial in analyzing the role of V1 neurons in motor circuits that generate movement of the limbs.

The conclusions of this paper are well supported by the data obtained using widely used methods. However, for some analyses, the specificity of labeling V1 clades should be clearly described.

(1) In Figure 1, the MafB antibody (Sigma) was used to identify Renshaw cells at P5. However, according to the supplementary Figure 3D, the specificity of the MafB antibody (Sigma) is relatively low. The image of MafB-GFP, V1-INs, and MafB-IR at P5 should be added to the supplementary figure. The specificity of MaFB-IR-Sigma in V1 neurons at P5 should be shown. This image also might support the description of the genetically labeled MafB-V1 distribution at P5 (page 8, lines 28-32).

(2) The proportion of genetically labeled FoxP2-V1 in all V1 is more than 60%, although immunolabeled FoxP2-V1 is approximately 30% at P5. Genetically labeled Otp-V1 included other non-FoxP2 V1 clades (Fig. 8L-M). I wonder whether genetically labeled FoxP2-V1 might include the other three clades. The authors should show whether genetically labeled FoxP2-V1 expresses other clade markers, such as pou6f2, sp8, and calbindin, at P5.

Reviewer #1 (Public Review):

Summary:

Bennion and colleagues present a careful examination of how an earlier set of memories can either interfere with or facilitate memories formed later. This impressive work is a companion piece to an earlier paper by Antony and colleagues (2022) in which a similar experimental design was used to examine how a later set of memories can either interfere with or facilitate memories formed earlier. This study makes contact with an experimental literature spanning 100 years, which is concerned with the nature of forgetting, and the ways in which memories for particular experiences can interact with other memories. These ideas are fundamental to modern theories of human memory, for example, paired-associate studies like this one are central to the theoretical idea that interference between memories is a much bigger contributor to forgetting than any sort of passive decay.

Strengths:

At the heart of the current investigation is a proposal made by Osgood in the 1940s regarding how paired associates are learned and remembered. In these experiments, one learns a pair of items, A-B (cue-target), and then later learns another pair that is related in some way, either A'-B (changing the cue, delta-cue), or A-B' (changing the target, delta-target), or A'-B' (changing both, delta-both), where the prime indicates that item has been modified, and may be semantically related to the original item. The authors refer to the critical to-be-remembered pairs as base pairs. Osgood proposed that when the changed item is very different from the original item there will be interference, and when the changed item is similar to the original item there will be facilitation. Osgood proposed a graphical depiction of his theory in which performance was summarized as a surface, with one axis indicating changes to the cue item of a pair and the other indicating changes to the target item, and the surface itself necessary to visualize the consequences of changing both.

In the decades since Osgood's proposal, there have been many studies examining slivers of the proposal, e.g., just changing targets in one experiment, just changing cues in another experiment. Because any pair of experiments uses different methods, this has made it difficult to draw clear conclusions about the effects of particular manipulations.

The current paper is a potential landmark, in that the authors manipulate multiple fundamental experimental characteristics using the same general experimental design. Importantly, they manipulate the semantic relatedness of the changed item to the original item, the delay between the study experience and the test, and which aspect of the pair is changed. Furthermore, they include both a positive control condition (where the exact same pair is studied twice), and a negative control condition (where a pair is only studied once, in the same phase as the critical base pairs). This allows them to determine when the prior learning exhibits an interfering effect relative to the negative control condition and also allows them to determine how close any facilitative effects come to matching the positive control.

The results are interpreted in terms of a set of existing theories, most prominently the memory-for-change framework, which proposes a mechanism (recursive reminding) potentially responsible for the facilitative effects examined here. One of the central results is the finding that a stronger semantic relationship between a base pair and an earlier pair has a facilitative effect on both the rate of learning of the base pair and the durability of the memory for the base pair. This is consistent with the memory-for-change framework, which proposes that this semantic relationship prompts retrieval of the earlier pair, and the two pairs are integrated into a common memory structure that contains information about which pair was studied in which phase of the experiment. When semantic relatedness is lower, they more often show interference effects, with the idea being that competition between the stored memories makes it more difficult to remember the base pair.

This work represents a major methodological and empirical advance for our understanding of paired-associates learning, and it sets a laudably high bar for future work seeking to extend this knowledge further. By manipulating so many factors within one set of experiments, it fills a gap in the prior literature regarding the cognitive validity of an 80-year-old proposal by Osgood. The reader can see where the observed results match Osgood's theory and where they are inconclusive. This gives us insight, for example, into the necessity of including a long delay in one's experiment, to observe potential facilitative effects. This point is theoretically interesting, but it is also a boon for future methodological development, in that it establishes the experimental conditions necessary for examining one or another of these facilitation or interference effects more closely.

Weaknesses:

One minor weakness of the work is that the overarching theoretical framing does not necessarily specify the expected result for each and every one of the many effects examined. For example, with a narrower set of semantic associations being considered (all of which are relatively high associations) and a long delay, varying the semantic relatedness of the target item did not reliably affect the memorability of that pair. However, the same analysis showed a significant effect when the wider set of semantic associations was used. The positive result is consistent with the memory-for-change framework, but the null result isn't clearly informative to the theory. I call this a minor weakness because I think the value of this work will grow with time, as memory researchers and theorists use it as a benchmark for new theory development. For example, the data from these experiments will undoubtedly be used to develop and constrain a new generation of computational models of paired-associates learning.

Reviewer #1 (Public Review):

Summary/Strengths:

This manuscript describes a stimulating contribution to the field of human motor control. The complexity of control and learning is studied with a new task offering a myriad of possible coordination patterns. Findings are original and exemplify how baseline relationships determine learning.

Weaknesses:

A new task is presented: it is a thoughtful one, but because it is a new one, the manuscript section is filled with relatively new terms and acronyms that are not necessarily easy to rapidly understand.

First, some more thoughts may be devoted to the take-home message. In the title, I am not sure manipulating a stick with both hands is a key piece of information. Also, the authors appear to insist on the term 'implicit', and I wonder if it is a big deal in this manuscript and if all the necessary evidence appears in this study that control and adaptation are exclusively implicit. As there is no clear comparison between gradual and abrupt sessions, the authors may consider removing at least from the title and abstract the words 'implicit' and 'implicitly'. Most importantly, the authors may consider modifying the last sentence of the abstract to clearly provide the most substantial theoretical advance from this study.

It seems that a substantial finding is the 'constraint' imposed by baseline control laws on sensorimotor adaptation. This seems to echo and extend previous work of Wu, Smith et al. (Nat Neurosci, 2014): their findings, which were not necessarily always replicated, suggested that the more participants were variable in baseline, the better they adapted to a systematic perturbation. The authors may study whether residual errors are smaller or adaptation is faster for individuals with larger motor variability in baseline. Unfortunately, the authors do not present the classic time course of sensorimotor adaptation in any experiment. The adaptation is not described as typically done: the authors should thus show the changes in tip movement direction and stick-tilt angle across trials, and highlight any significant difference between baseline, early adaptation, and late adaptation, for instance. I also wonder why the authors did not include a few no-perturbation trials after the exposure phase to study after-effects in the study design: it looks like a missed opportunity here. Overall, I think that showing the time course of adaptation is necessary for the present study to provide a more comprehensive understanding of that new task, and to re-explore the role of motor variability during baseline for sensorimotor adaptation.

The distance between hands was fixed at 15 cm with the Kinarm instead of a mechanical constraint. I wonder how much this distance varied and more importantly whether from that analysis or a force analysis, the authors could determine whether one hand led the other one in the adaptation.

I understand the distinction between task- and end-effector irrelevant perturbation, and at the same time results show that the nervous system reacts to both types of perturbation, indicating that they both seem relevant or important. In line 32, the errors mentioned at the end of the sentence suggest that adaptation is in fact maladaptive. I think the authors may extend the Discussion on why adaptation was found in the experiments with end-effector irrelevant and especially how an internal (forward) model or a pair of internal (forward) models may be used to predict both the visual and the somatosensory consequences of the motor commands.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Liangliang Fu and colleagues propose that a population of CD81-positive fibroblasts exhibiting senescent features activate neutrophils via the C3/C3aR1 axis and contribute to maintaining the inflammatory response in periodontitis. The authors provide evidence that inhibition of cellular senescence by metformin treatment in murine models ameliorated periodontitis progression. This study provides some valuable insights into the impact of periodontitis-induced gingival damage and the significance of stromal senescence.

Strengths:

(1) The work combines a variety of models of periodontitis, including analyses of human samples, primary gingival fibroblast cell culture isolation and cultures, and mouse models of ligature/induced periodontitis. Then, the results are solid in terms of used models.

(2) Comprehensive exhibition of methodologies incorporating histology procedures, micro-CT imaging, bulkRNAseq and scRNAseq transcriptomic profiles (the latter analyses of published datasets), and a number of computational analyses. The paper is robust at the technical level.

(3) This paper is timely and interesting and it opens potential therapeutic avenues for the treatment of periodontitis. Although the interplay of senescence with periodontitis and the use of metformin has been previously reported (e.g. Kuang et al. Biogerontology 2020), I think the proposed mechanism of neutrophils activation by CD81-positive senescent fibroblasts and the inflammatory response is original. The paper is therefore at the forefront of the field, as senescence and its interplay with the immune system is a hot topic and reflects the current directions ("trending topics") of the field.

Weaknesses:

(1) The assessment of Cellular Senescence is limited and would benefit from additional biomarkers and not just p16 and p21, in particular in vivo.

(2) This paper does not include original scRNAseq datasets in periodontitis, but analyses of already published datasets.

(3) The authors claim that cellular senescence of CD81+ fibroblasts could be attributed to disturbances of lipid metabolism, resulting in differentiation arrest and higher expression of SASP factors in CD81+ fibroblast cells. Although the authors found that a series of pathways related to metabolism (metabolism of linoleic acid, linolenic acid, arachidonic acid, or steroid biosynthesis) are upregulated in CD81+ fibroblasts by transcriptomic analyses the hypothesis remains speculative and requires further validations.

(4) Metformin has been reported to downregulate the SASP and lower senescent cell burden (e.g. for review see Kulkarni, Gubbi, and Barzilai. Cell Metab 2020). Although Metformin's senotherapeutic activities can be mediated by anti-inflammatory effects preventing NFkB translocation to the nucleus (Moiseeva et al. Aging Cell 2013) and has been shown to prevent oxidative stress-induce senescence in human periodontal ligament cells (Kuang et al. Biogerontology 2020) it can also drive multiple and pleiotropic effects unrelated to senescence.

(5) Mechanistically, the proposed activation neutrophils by senescent C81+ fibroblasts via the C3/C3aR1 axis would be further supported by using a senolytic approach (e.g. Bcl2 inhibitor) allowing testing of whether eradication of senescent stromal cells results in reduced levels of CD81 and C3 positivity, and prevention of neutrophils infiltration.

Reviewer #1 (Public Review):

Summary:

De Waele et al. reported a dual-branch neural network model for predicting antibiotic resistance profiles using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry data. Neural networks were trained on the recently available DRIAMS database of MALDI-TOF mass spectrometry data and their associated antibiotic susceptibility profiles. The authors used a dual branch neural network approach to simultaneously represent information about mass spectra and antibiotics for a wide range of species and antibiotic combinations. The authors showed consistent performance of their strategy to predict antibiotic susceptibility for different spectrums and antibiotic representations (i.e., embedders). Remarkably, the authors showed how small datasets collected at one location can improve the performance of a model trained with limited data collected at a second location. Despite these promising results, there are several analyses that the authors could incorporate to offer additional support to some of their claims (see weaknesses). In particular, this work would benefit from a more comprehensive comparison of the author's single recommender model vs an ensemble of specialist models, and the inclusion of 1-2 examples that showcase how their model could be translated into the clinic.

Strengths:

• A single AMR recommender system could potentially facilitate the adoption of MALDI-TOF-based antibiotic susceptibility profiling into clinical practices by reducing the number of models to be considered, and the efforts that may be required to periodically update them.

• Authors tested multiple combinations of embedders for the mass spectra and antibiotics while using different metrics to evaluate the performance of the resulting models. Models trained using different spectrum embedder-antibiotic embedder combinations had remarkably good performance for all tested metrics. The average ROC AUC scores for global and spectrum-level evaluations were above 0.9. Average ROC AUC scores for antibiotic-level evaluations were greater than 0.75.

• Authors showed that data collected in one location can be leveraged to improve the performance of models generated using a smaller number of samples collected at a different location. This result may encourage researchers to optimize data integration to reduce the burden of data generation for institutions interested in testing this method.

Weaknesses:

• Although ROC AUC is a widely used metric. Other metrics such as precision, recall, sensitivity, and specificity are not reported in this work. The last two metrics would help readers understand the model's potential implications in the context of clinical research.

• The authors did not hypothesize or describe in any way what an acceptable performance of their recommender system should be in order to be adopted by clinicians.

• Related to the previous comment, this work would strongly benefit from the inclusion of 1-2 real-life applications of their method that could showcase the benefits of their strategy for designing antibiotic treatment in a clinical setting.

• The authors do not offer information about the model features associated with resistance. This information may offer insights about mechanisms of antimicrobial resistance and how conserved they are across species.

• Comparison of AUC values across models lacks information regarding statistical significance. Without this information it is hard for a reader to figure out which differences are marginal and which ones are meaningful (for example, it is unclear if a difference in average AUC of 0.02 is significant). This applied to Figure 2, Figure 3, and Table 2 (and the associated supplementary figures).

• One key claim of this work was that their single recommender system outperformed specialist (single species-antibiotic) models. However, in its current status, it is not possible to determine that in fact that is the case (see comment above). Moreover, comparisons to species-level models (that combine all data and antibiotic susceptibility profiles for a given species) would help to illustrate the putative advantages of the dual branch neural network model over species-based models. This analysis will also inform the species (and perhaps datasets) for which specialist models would be useful to consider.

• Taking into account that the clustering of spectra embeddings seemed to be species-driven (Figure 4), one may hypothesize that there is limited transfer of information between species, and therefore the neural network model may be working as an ensemble of species models. Thus, this work would deeply benefit from a comparison between the authors' general model and an ensemble model in which the species is first identified and then the relevant species recommender is applied. If authors had identified cases to illustrate how data from one species positively influence the results for another species, they should include some of those examples.

Reviewer #1 (Public Review):

Summary:

This work extends previous agent-based models of murine muscle regeneration by the authors (especially Westman et al., 2021) and by others (especially Khuu et al, 2023) by incorporating additional agent rules (altogether now based on over 100 published studies), threshold parameters and interactions with fields of cytokines and growth factors as well as capillaries (dynamically changing through damage and angiogenesis) and lymphatic vessels. The estimation of 52 unknown parameters against three time courses of tissue-scale observables (muscle cross-sectional area recovery, satellite stem cell count and fibroblast cell count) employs the CaliPro algorithm (Joslyn et al., 2021) and sensitivity analysis. The model is validated against additional time courses of tissue-scale observables and qualitative perturbation data, which match almost all conditions. This model is here used to predict (also non-monotonic) responses of (combinations of) cytokine perturbations but it moreover represents a valuable resource for further analysis of emergent behavior across multiple spatial scales in a physiologically relevant system.

Strengths:

This work (almost didactically) demonstrates how to develop, calibrate, validate and analyze a comprehensive, spatially resolved, dynamical, multicellular model. Testable model predictions of (also non-monotonic) emergent behaviors are derived and discussed. The computational model is based on a widely-used simulation platform and shared openly such that it can be further analyzed and refined by the community. The single-used parameter set is a good starting point for future work that can, as outlined in the discussion section of the paper, analyze model results from the full distribution of matching parameter values and for a spectrum of realistic tissue configurations.

Reviewer #1 (Public Review):

Summary:

In this study, Komarova et al. investigate the clinical prognostic ability of cell-level metabolic heterogeneity quantified via the fluorescence lifetime characteristics of NAD(P)H. Fluorescence lifetime imaging microscopy (FLIM) has been studied as a minimally invasive approach to measure cellular metabolism in live cell cultures, organoids, and animal models. Its clinical translation is spearheaded through macroscopic implementation approaches that are capable of large sampling areas and enable access to otherwise constrained spaces but lack cellular resolution for a one-to-one transition with traditional microscopy approaches, making the interpretation of the results a complicated task. The merit of this study primarily lies in its design by analyzing with the same instrumentation and approach colorectal samples in different research scenarios, namely in vitro cells, in vivo animal xenografts, and tumor tissue from human patients. These conform to a valuable dataset to explore the translational interpretation hurdles with samples of increasing levels of complexity. For human samples, the study specifically investigates the prediction ability of NAD(P)H fluorescence metrics for the binary classification of tumors of low and advanced stage, with and without metastasis, and low and high grade. They find that NAD(P)H fluorescence properties have a strong potential to distinguish between high- and low-grade tumors and a moderate ability to distinguish advanced-stage tumors from low-stage tumors. This study provides valuable results contributing to the deployment of minimally invasive optical imaging techniques to quantify tumor properties and potentially migrate into tools for human tumor characterization and clinical diagnosis.

Strengths:

The investigation of colorectal samples under multiple imaging scenarios with the same instrument and approach conforms to a valuable dataset that can facilitate the interpretation of results across the spectrum of sample complexity.

The manuscript provides a strong discussion reviewing studies that investigated cellular metabolism with FLIM and the metabolic heterogeneity of colorectal cancer in general.

The authors do a thorough acknowledgement of the experimental limitations of investigating human samples ex vivo, and the analytical limitation of manual segmentation, for which they provide a path forward for higher throughput analysis.

Weaknesses:

To substantiate the changes in fluorescence properties at the examined wavelength range (associated with NAD(P)H fluorescence) in relationship to metabolism, the study would strongly benefit from additional quantification of metabolic-associated metrics using currently established standard methods. This is especially interesting when discussing heterogeneity, which is presumably high within and between patients with colorectal cancer, and could help explain the particularities of each sample leading to a more in-depth analysis of the acquired valuable dataset. Additionally, NAD(P)H fluorescence does not provide a complete picture of the cell/tissue metabolic characteristics. Including, or discussing the implications of including fluorescence from flavins would comprise a more compelling dataset. These additional data would also enable the quantification of redox metrics, as briefly mentioned, which could positively contribute to the prognosis potential of metabolic heterogeneity.

In the current form of the manuscript, there is a diluted interpretation and discussion of the results obtained from the random forest and SHAP analysis regarding the ability of the FLIM parameters to predict clinicopathological outcomes. This is, not only the main point the authors are trying to convey given the title and the stated goals, but also a novel result given the scarce availability of these type of data, which could have a remarkable impact on colorectal cancer in situ diagnosis and therapy monitoring. These data merit a more in-depth analysis of the different factors involved. In this context, the authors should clarify how is the "trend of association" quantified (lines 194 and 199).

Reviewer #1 (Public Review):

Summary:

The authors propose an improved neuro-muscle co-culture system to study ALS-related functional differences in human pluripotent stem cell lines.

Strengths:

A simple co-culture system with functional readout.

Weaknesses:

There are concerns about the lack of novelty, rigor, and clarity in the approach. The strength of the study is undermined by its reliance on transcription factors used more than a decade ago, low myocyte activity, and inadequate validation methods, such as the lack of single-cell transcriptome analysis and detailed neuromuscular synapse characterization. The evidence presented requires substantial validation through rigorous experimental approaches and resolution of the identified concerns for the study's findings to be considered significant and reliable.

for - sleep hygiene - 1 - regularity

advice - sleep hygiene - 1 - regularity - try to make weekend sleep times same as weekday - don't deviate if possible

Reviewer #1 (Public Review):

Summary:

The authors collected genomic information from public sources covering 423 eukaryote genomes and around 650 prokaryote genomes. Based on pre-computed CDS annotation, they estimated the frequency of alternative splicing (AS) as a single average measure for each genome and computed correlations with this measure and other genomic properties such as genome size, percentage of coding DNA, gene and intergenic span, etc. They conclude that AS frequency increases with genome complexity in a somewhat directional trend from "lower" organisms to "higher" organisms.

Strengths:

The study covers a wide range of taxonomic groups, both in prokaryotes and eukaryotes.

Weaknesses:

The study is weak both methodologically and conceptually. Current high throughput sequencing technologies, coupled with highly heterogeneous annotation methods, can observe cases of AS with great sensitivity, and one should be extremely cautious of the biases and rates of false positives associated with these methods. These issues are not addressed in the manuscript. Here, AS measures seem to be derived directly from CDS annotations downloaded from public databases, and do not account for differing annotation methods or RNA sequencing depth and tissue sample diversity.

There is no mention of the possibility that AS could be largely caused by random splicing errors, a possibility that could very well fit with the manuscript's data. Instead, the authors adopt early on the view that AS is regulated and functional, generally citing outdated literature.

There is no question that some AS events are functional, as evidenced by strongly supported studies. However, whether all AS events are functional is questionable, and the relative fractions of functional and non-functional AS are unknown. With this in mind, the authors should be more cautious in interpreting their data. The "complexity" of organisms also correlates well (negatively) with effective population size. The power of selection to eliminate (slightly) deleterious mutations or errors decreases with effective population size. The correlation observed by the authors could thus easily be explained by a non-adaptive interpretation based on simple population genetics principles.

The manuscript contains evidence that the authors might benefit from adopting a more modern view of how evolution proceeds. Sentences such as "... suggests that only sophisticated organisms optimize alternative splicing by increasing..." (L113), or "especially in highly evolved groups such as mammals" (L130), or the repeated use of "higher" and "lower" organisms need revising.

Because of the lack of controls mentioned above, and because of the absence of discussion regarding an alternative non-adaptive interpretation, the analyses presented in the manuscript are of very limited use to other researchers in the field. In conclusion, the study does not present solid conclusions.

Reviewer #1 (Public Review):<br /> <br /> Summary:

This paper provides a methodology for normative trajectory modeling, using cross-sectional data to set the "norms," and then applying these norms to longitudinal brain observations. An example of schizophrenia trajectories (two time points) is provided. The method assumes a Bayesian mixed effects model, which included some hyperparameters that need to be tuned. The longitudinal assumption is essentially a random intercept model, assuming that the age-based quantiles do not shift, and if they do that is a sign of disease-like trajectories.

Strengths:

Normative modeling of brain feature trajectories is an important topic. Bayesian models are a promising alternative to modeling these. Leveraging large-scale data to provide norms is also potentially useful.

Weaknesses:

The models described are not fundamentally novel, essentially a random intercept model (with a warping function), and some flexible covariate effects using splines (i.e., additive models). The assumption of constant quantiles is very strong, and limits the utility of the model to very short term data. The schizophrenia example leads to a counter-intuitive normalization of trajectories, which leads to suspicions that this is driven by some artifact of the data modeling/imaging pipelines. The method also assumes that the cross-sectional data is from a "healthy population" without describing what this population is (there is certainly every chance of ascertainment bias in large scale studies as well as small scale studies). This issue is completely elided over in the manuscript.

Reviewer #1 (Public Review):

The authors sought to determine the impact of early antiretroviral treatment on the size, composition, and decay of the HIV latent reservoir. This reservoir represents the source of viral rebound upon treatment interruption and therefore constitutes the greatest challenge to achieving an HIV cure. A particular strength of this study is that it reports on reservoir characteristics in African women, a significantly understudied population, of whom some have initiated treatment within days of acute HIV diagnosis. With the use of highly sensitive and current technologies, including digital droplet PCR and near full-length genome next-generation sequencing, the authors generated a valuable dataset for investigation of proviral dynamics in women initiating early treatment compared to those initiating treatment in chronic infection. The authors confirm previous reports that early antiretroviral treatment restricts reservoir size, but further show that this restriction extends to defective viral genomes, where late treatment initiation was associated with a greater frequency of defective genomes. Furthermore, an additional strength of this study is the longitudinal comparison of viral dynamics post-treatment, wherein early treatment was shown to be associated with a more rapid rate of decay in proviral genomes, regardless of intactness, over a period of one year post-treatment. While it is indicated that intact genomes were not detected after one year following early treatment initiation, caution should be taken with interpretation where sequence numbers are low. Defective genomes are more abundant than intact genomes and are therefore more likely to be sampled. Early treatment was also associated with reduced proviral diversity and fewer instances of polymorphisms associated with cytotoxic T-lymphocyte immune selection. This is expected given that rapid evolution and extensive immune selection are synonymous with HIV infection in the absence of treatment, yet points to an additional benefit of early treatment in the context of immune therapies to restrict the reservoir.

Given that this is one of the first studies to report the mapping of longitudinal intactness of proviral genomes in the globally dominant subtype C, the manuscript would benefit from placing these findings in the context of what has been reported in other populations, for example, how decay rates of intact and defective genomes compare with that of other subtypes where known. While not a primary outcome of the study, the comparisons of peak viremia in the hyperacute and chronic-treated groups may be confounded by the fact that peak viremia may have been pre-empted by early treatment i.e., the true peak was not reached in early-treated individuals. Indeed, in the abstract, the authors indicate that treatment was initiated before the peak. The use of the term 'peak' viremia in the hyperacute-treated group could perhaps be replaced with 'highest recorded viral load'. The statistical comparison of this measure in the two groups is perhaps more relevant with regards to viral burden over time or area under the curve viral load as these are previously reported as correlates of reservoir size. The analysis of clonal expansion of proviral genomes may be limited by higher sequence homogeneity in hyperacute infection i.e., cells with different proviral integration sites may have a higher likelihood of containing identical genomes than chronic infection.

Overall, these data demonstrate the distinct benefits of early treatment initiation at reducing the barrier to a functional cure for HIV, not only by restricting viral abundance and diversity but also potentially through the preservation of immune function and limiting immune escape. It therefore provides clues to curative strategies even in settings where early diagnosis and treatment may be unlikely.